Biochemical bases in differentiation of a mouse cell line GSM06 to gastric surface cells.

A mouse gastric surface cell line GSM06 established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene was subjected to the lipid and glycoprotein analysis. When GSM06 cells were cultured for a long time after formation of a confluent monolayer, they differentiated to resemble foveolar epithelial cells morphologically. Biochemical changes during culture were studied in cells harvested just when a monolayer had formed (day 0), on day 7, and on day 21. Content of total phospholipids, cholesterol, cholesterol sulfate, total sugar and sialic acid increased about 1.5-fold from day 0 to 7 and remained elevated till day 21. The fatty acid composition of phospholipids revealed increased relative levels of oleic acid in phosphatidylcholine and phosphatidylethanolamine, and an increased level of plasmenylethanolamine from day 0 to 7. The level of dolichylphosphate continued to increase in a time-dependent manner. Glycosylation of various proteins, detected with lectins, was enhanced from day 7. In addition, greater resistance to taurodeoxycholate and acetylsalicylic acid was observed on days 7 and 21 than on day 0. Thus, enhanced glycosylation of proteins and an overall increase in the area of cellular membranes were the major changes in GSM06 cells during culture, and they were accompanied by an enhancement of cytoprotective potential.

Potential usage of thermosensitive liposomes for site-specific delivery of cytokines.

Long-circulating liposomes reside long in the bloodstream, and transient swelling during phase transition of liposomes with hyper-osmotic internal aqueous phase causes release of macromolecules. Here we examined the applicability of long-circulating thermosensitive liposomes for delivery of tumor necrosis factor (TNF) by the heating of a local tumor-growing site after injection of TNF-loaded liposomes into tumor-bearing mice. Glucuronate modified thermosensitive liposomes with internal solution of two-fold higher osmotic pressure and sized through 200 nm-pore, released encapsulated [(131)I] human serum albumin at 42 degrees C in vitro and showed long-circulating character in vivo. Cytotoxic action of TNF encapsulated in long-circulating thermosensitive-liposomes (LCTS-liposomes) against L929 fibrosarcoma cells was enhanced at 42 degrees C in vitro. Furthermore, the tumor growth tended to be inhibited more by hyperthermia of mice bearing Meth A sarcoma than without heating after injection of TNF encapsulated in LCTS-liposomes. These results suggest that the cytokine can be released at the tumor site from the circulating CLTS-liposomes.

Detection of p53 gene mutations in fine-needle aspiration biopsied breast cancer specimens: correlations with nuclear p53 accumulations and tumor DNA aneuploidy patterns.

Using the polymerase chain reaction and a single strand conformation polymorphism (PCR-SSCP) analysis, p53 gene mutations have been examined preoperatively in aspirated biopsy specimens from 22 breast cancers. In eight (36%) of the studied cases, abnormal bands indicating mutations were detected and were more frequently present in common invasive breast carcinomas. Eight of the breast cancer cases were found to be positive for mutations: direct DNA sequencing confirmed eight mutations and revealed G to A and A to G nucleotide changes in five (63%) and two (25%) cases, respectively. The mutations that were localized at the CpG site of the gene were seen in three cases. Additionally, an immunohistochemical investigation of the presence of the p53 protein and a DNA flow-cytometrical analysis of the 22 resected breast cancers were also performed. Nuclear p53 protein accumulations and a DNA aneuploidy pattern were detected in 11 (50%) cases and in 16 (73%) cases, respectively, and both significantly correlated with p53 gene mutations (P < 0.01 and P < 0.001, respectively; Fisher's exact test). In contrast, in five cases of a breast cancer with a lower histologic grade, no p53 gene mutations, nuclear p53 protein accumulations, or DNA aneuploidy patterns were detected. These results thus suggest that p53 gene mutations, nuclear accumulations of the p53 protein, and a DNA aneuploidy pattern are events that occur with close relationship in the progression of a breast cancer, and that p53 abnormalities appear to correlate with a high grade of the malignancy. Therefore, the detection of p53 gene mutations in aspirated biopsy specimens of breast cancers may be helpful for an accurate and rapid cytological diagnosis, which reflects the prognosis.

Postnatal change of pig intestinal ganglioside bound by Escherichia coli with K99 fimbriae.

Enterotoxigenic Escherichia coli possessing K99 fimbriae (E. coli K99) causes diarrhea in piglets of less than 1 week old. The first stage of the bacterial infection is adhesion by the fimbriae on the small intestinal mucosa and the adhesion is followed by colony formation. K99 fimbriae bind specifically to N-glycolylneuraminyl-lactosyl-ceramide, GM3(NeuGc) [Ono, E. et al. (1989) Infect. Immun. 57,907-911]. We examined the postnatal change of the content and the molecular species of GM3(NeuGc) in the small intestinal mucosa of 0- to 14-day-old piglets and adult pigs. GM3(NeuGc) was a major ganglioside of piglet intestinal mucosa. GM3(NeuGc) content was maximal at birth and gradually decreased to 1/16 in adult animals (5 months old). The ceramide moiety of piglet intestinal GM3(NeuGc) was characterized by the presence of 2-hydroxylated palmitic acid. 125I-labeled bacteria strongly bound to GM3(NeuGc) containing 2-hydroxylated palmitic acid and phytosphingosine compared with GM3(NeuGc) containing any other ceramide moiety. The time when this particular GM3(NeuGc) appears coincides with the time that the infection occurs, and it may explain the susceptibility of newborn piglets to E. coli K99 infection.

Neoadjuvant intra-arterial infusion chemotherapy combined with hormonal therapy for locally advanced breast cancer.

To determine the efficacy of combined neoadjuvant intra-arterial infusion chemotherapy and hormonal therapy for treating locally advanced breast cancer, we compared the outcomes of patients with or without this therapy, and also assessed histologic response. Ninety-four patients with locally advanced breast cancer (stage IIIa, 56; stage IIIb, 38). Nineteen stage IIIa and 17 stage IIIb patients received intra-arterial plus hormonal therapy while 37 stage IIIa and 21 stage IIIb patients with similar ages and follow-up durations did not. Treated patients received intra-arterial epirubicin plus oral medroxy-progesterone. Five-year disease-free survival rates were 77.5% for intra-arterially treated and 33.0% for other patients in stage IIIa, and 70.5% for intra-arterially treated and 38.1% for other patients in stage IIIb. Five-year overall survival rates were 94.4% for intra-arterially treated and 61.7% for other patients in stage IIIa, and 90.9% for intra-arterially treated and 56.3% for other patients in stage IIIb. Ten-year overall survival rates in stage IIIb were 90.9% for treated and 22.5% for other group patents. All differences were statistically significant (p<0.05). Good histologic response to intra-arterial therapy was seen in 75% of the primary tumors and 71% of involved lymph nodes. Neoadjuvant intra-arterial therapy with hormonal therapy yielded better survival rates than no intra-arterial therapy or our previous intra-arterial regimen.

Neoadjuvant intraarterial high-dose chemotherapy and autologous peripheral blood stem cell transplantation for advanced breast cancer.

Eight locally advanced breast cancer patients were treated with neoadjuvant intraarterial high-dose chemotherapy (epirubicin) plus MPA combined with autologous PBSCT. All patients completed the scheduled treatment, and there were no toxic deaths. Patients were treated with an escalating dose of epirubicin (370-480 mg) and cyclophosphamide (0-6000 mg). The rate of clinical response was 100%. The rate of good histologic response was 87.5% in the main tumor and 75% in diseased lymph nodes. The 2-year survival rate was 100%. Six patients were disease-free at the time of writing. This treatment resulted in higher rates of clinical and histologic response when compared with standard-dose intraarterial chemotherapy.

Unusual complications caused by endo-clip migration following a laparoscopic cholecystectomy: report of a case.

We present herein the case of a woman who developed unexpected complications following a laparoscopic cholecystectomy. The clinical manifestations were unique in that symptoms of obstructive jaundice were in fact caused by endo-clips which had migrated into the common bile duct, and the resulting choledochoduodenal fistula presented as persistent cholangitis. While the overall safety of this new procedure appears to be superior to that of open cholecystectomy, the risk of bile duct injury seems more likely. This case stresses the need for caution by surgeons, and emphasizes the necessity for careful surveillance and strict follow-up to ensure the safety of this procedure.

Cloning and expression of the human 5-HT1B-type receptor gene.

We report the cloning and expression of a novel 5-HT receptor gene from human genomic DNA. This clone, HGCR1, contains an apparently intronless open reading frame of 390 amino acids with the seven hydrophobic regions, typical of G-protein coupled receptors. The deduced amino acid sequence of HGCR1 is 39%, 55% and 87% identical to that for the human 5-HT1A, the human 5-HT1D and the rat 5-HT1B receptor, respectively. [3H]5-HT binding to transfected COS-7 cell membranes yields a pharmacological profile similar to that of 5-HT1B receptor. Thus these findings indicate the presence of 5-HT1B-type receptor in the human.

Detection of N-acetylgalactosaminyltransferase mRNA which determines expression of Sda blood group carbohydrate structure in human gastrointestinal mucosa and cancer.

The Sda blood group carbohydrate structure, GalNAcbeta1-4[NeuAcalpha2-3]Galbeta1-4GlcNAc-R, is expressed on glycolipid and glycoprotein in human gastrointestinal mucosa. The expression of the Sda determinant dramatically decreases in cancer tissue. The activity of the beta1,4N-acetylgalactosaminyltransferase (Sda-GalNAcT), which transfers GalNAc to NeuAcalpha2-3Galbeta1-4Glc(NAc)-R, correlates with the expression of the Sda immuno-epitope. From the total RNA fraction of human gastric mucosa, we have amplified a cDNA segment by reverse-transcription-polymerase-chain reaction (RT-PCR), using primers designed according to the cDNA sequence of a murine beta1,4GalNAcT which synthesizes the Sda determinant. An RT-PCR product of 390 bp shared 85% nucleotide identity with the murine Sda-related beta1,4GalNAcT. This RT-PCR product hybridized to a transcript in mRNA prepared from human gastric mucosa. In RT-PCR using specific primers to this PCR product, Sda-GalNAcT mRNA was detected in all samples of normal stomach and small intestine examined and the majority of normal colonic specimens. Six out of nine cases of gastric cancer, and 9 out of 13 cases of colonic cancer failed to produce the target DNA. These results correlate with the beta1,4GalNAcT activity measured in the same samples. In conclusion, a segment of the cDNA for betal,4GalNAcT which determines expression of the Sda carbohydrate structure was obtained, and reduced transcription of this beta 1,4GalNAcT resulted in the disappearance of the Sda epitope in gastrointestinal cancer.

Enhanced expression of GM2/GD2 synthase mRNA in human gastrointestinal cancer.

BACKGROUND: The content of the GM2 ganglioside and the activity of UDP-GalNAc: GM3 beta-1,4N-acetylgalactosaminyltransferase (beta-1,4GalNAcT), which synthesizes GM2, increased in gastric cancer tissues and gastric cancer cell lines as compared with that in normal gastric mucosa. METHODS: Expression of beta-1,4GalNAcT mRNA and a concentration of GM2 in the human gastrointestinal tissues were examined. Beta-1,4GalNAcT mRNA in human surgical specimens, which was not detectable with Northern blotting because of the paucity of absolute amounts expressed, was detected with competitive reverse transcription-polymerase chain reaction (PCR) method using an internal standard cRNA that could be amplified by the same primers as target mRNA in PCR. The quantification of GM2 was examined using immunostaining of thin-layer chromatography. RESULTS: In 10 of 10 gastric carcinomas and 6 of 13 colonic carcinomas, mRNA expression was more enhanced than that in the normal mucosa of each patient. The alteration of GM2 content in carcinoma from normal tissue generally was correlated to the change in the expression of beta-1,4GalNAcT mRNA with a few exceptions. One gastric cancer sample had a higher level of mRNA with a lower GM2 content than the corresponding normal tissue, and two colonic carcinoma tissue specimens had a lower level of mRNA with a higher GM2 content. CONCLUSIONS: These results suggest that expression of the beta-1,4GalNAcT gene is a key step in the molecular mechanisms underlying the regulation of cancer-associated GM2 expression in the stomach and the colon.

Influence of tooth-to-denture-base discrepancy on space closure following premature loss of deciduous teeth.

Influence of tooth-to-denture-base discrepancy on so-called physiologic migration of the first molar was studied on serial dental casts of 116 boys and girls, obtained through a dental health program for school children in an area in which there was no dentist. The alteration of spaces following premature loss of deciduous molars was examined comparing the anterior to posterior discrepancies between tooth and denture base. Modes of space alteration showed positive correlation with the size of the discrepancy, especially in the mandibular dental arches. The space deficiency in the posterior region seemed to have a positive effect on the mesial migration of the first molar. Mesial migration of the first molar seems to be pathologic rather than physiologic and is strongly affected by tooth-to-denture-base discrepancies. Space maintenance does not seem to be useful, because it is not necessary in minimum discrepancy cases and is not effective in severe discrepancy cases.

Ileal duplication cyst associated with heterotopic pancreas: report of a case and literature review.

A rare case of solitary ileal duplication cyst accompanied by heterotopic pancreas in the terminal ileum is presented. An 8 year old boy with an ovoid shaped, elastic soft and pendant-growing ileal duplication cyst associated with aberrant pancreatic tissue but not communicating with the lumen of the ileum, underwent an adhesiotomy for a small intestinal adhesion that caused bowel obstruction. The obstructive state of the intestine seemed to be caused by an inflammation of the duplication cyst which was 4.5 x 2.7 x 2.5 cm and oval in shape. Excision of the cyst and the heterotopic pancreas was curative. The histological findings were identical to heterotopic pancreas accompanied by ileal duplication without any clinical features of heterotopic pancreas. A review of the literature is presented along with the author's experience in a case of ileal duplication cyst associated with heterotopic pancreas located in the ileum.