Polarographic detection of reoxygenation of lymph node metastases in the initial phase of primary radio- and radiochemotherapy in patients with advanced squamous epithelial carcinomas of the head and neck

Tyrosine phosphorylation is widely recognized as playing an important role in cell differentiation, proliferation and carcinogenesis. We used the polymerase chain reaction (PCR) method to identify protein tyrosine kinases that were expressed in the skin. Mixed oligonucleotide probes were used to amplify and screen neonatal murine skin mRNA for clones encoding amino acid contiguities, the conservation of which is characteristic of the protein tyrosine kinase family. When the PCR products were sequenced, a novel clone encoding protein tyrosine kinase, PTK70, was identified. A full-length cDNA was isolated from a mouse thymus cDNA library. The nucleotide and deduced amino acid sequence showed that it featured src-homology (SH) 2 domain, SH3 domain and kinase domain like other src family protein tyrosine kinases, but lacked the N-terminal myristylation site and C-terminal tyrosine residue. Although the mRNA of PTK70 was detected in various tissues ubiquitously, the degree of its expression differed among tissues. Murine skin is one in which PTK70 was expressed strongly, with its expression being much stronger in the epidermis and in the cell line derived from murine keratinocytes than in those from melanoma or fibroblast cell lines. These evidences suggest that PTK70 may be involved in proliferation or differentiation of keratinocytes in the skin.     

Uses of nitric oxide in obstetrics and gynecology

A cDNA clone encoding a human ribosomal protein L39 (hRPL39) was isolated through a random cDNA sequencing approach to a cDNA library constructed from a human colon carcinoma cell line of COLO 205. Although levels of hRPL39 mRNA were different in several cell lines including carcinoma cell lines from different tissues, they were shown not to be cell cycle-dependent in a human fibroblast cell line of TIG-1.     

Aerobic growth and survival of Campylobacter jejuni in food and stream water

OBJECTIVE: To search differentially expressed sequences correlated with pathogenesis of human nasopharyngeal carcinoma (NPC), including the candidates of tumor suppressor genes. METHODS: cDNA representational difference analysis (RDA) was performed to isolate differentially expressed sequences between cDNA from normal human primary cultures of nasopharyngeal epithelial cells and cDNA from NPC cell line HNE1. The sources of differentially expressed products were proved by Southern blot and Northern blot. The fragments were cloned with pGEM-T easy kit and sequenced by the chain termination reaction. RESULTS: Four differentially expressed cDNA fragments were isolated in the fourth subtractive hybridization using cDNA from normal human primary cultures of nasopharyngeal epithelial cells as tester amplicon and cDNA from NPC cell line HNE1 as driver amplicon by cDNA RDA. These differential cDNA fragments revealed that they really came from the tester amplicon and were not expressed or down-regulated in the NPC HNE1 cells. Of these obtained clones, some are the fragments of the human known genes including house-keeping genes, the others are novel genes. CONCLUSION: NPC involves alteration of multiple genes. Some of known genes matched with the differentially expressed sequences have an effective suppressive ability on the carcinoma.     

Identification of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing

A high-throughput direct-differential cDNA sequencing approach was employed to identify genes differentially expressed in normal breast as compared with breast cancer. Approximately 6000 expressed sequence tags (ESTs) from cDNA libraries of normal breast and breast carcinoma were selected randomly and subjected to EST-sequencing analysis. The relative expression levels of more than 2000 unique EST groups were quantitatively compared in normal versus cancerous breast. Of many putative differentially expressed genes, a breast cancer-specific gene, BCSGC1, which was expressed in high abundance in a breast cancer cDNA library but scarcely in a normal breast cDNA library, was identified as a putative breast cancer marker. In situ hybridization analysis demonstrated stage-specific BCSG1 expression as follows: BCSG1 was undetectable in normal or benign breast lesions, showed partial expression in ductal carcinoma in situ, but was expressed at an extremely high level in advanced infiltrating breast cancer. The predicted amino acid sequence of BCSG1 gene has a significant sequence homology to the non-amyloid beta protein fragment of the Alzheimer's disease amyloid protein. BCSG1 overexpression may indicate breast cancer malignant progression from benign breast or in situ carcinoma to the highly infiltrating carcinoma.     

Expression of structure-specific recognition protein mRNA in fetal kidney and Fe-nitrilotriacetate-induced renal carcinoma in the rat

Specific expression of the structure-specific recognition protein (SSRP) gene was investigated in rat fetal, adult, and tumor tissues using a 2.0-kb partial sequence of rat SSRP cDNA isolated from a cDNA library of rat renal cell carcinoma. The results revealed that it was rather specifically expressed in rat fetal kidney and renal cell carcinoma induced by Fenitrilotriacetate, but not in adult kidney, when various organs were tested by Northern blot analysis. In situ hybridization further demonstrated that it was located in the neoplastic cells of renal cell carcinoma and in the epithelial cells of fetal kidney but undetectable in any cells of normal adult kidney. These observations seem to imply the involvement of SSRP gene, which is believed to recognize structural alterations of DNA, in kidney development and carcinogenesis of certain types of kidney cancer.     

WAF1/p21 regulates proliferation, but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines

The WAF1/p21 gene product is an inhibitor of cyclin-dependent kinases which can be induced by the tumor suppressor p53 and mediate some of its effects, or function in p53-independent pathways of cell cycle regulation. Although a potential tumor suppressor gene, WAF1/p21 is expressed in bladder cancer. To elucidate the function of p21 in tumor cells we have investigated in urothelial carcinoma cell lines: i) WAF1/p21 mRNA and protein expression, ii) the biological effects of p21 overexpression or down-regulation and (iii) whether p21 can be induced by p53. WAF1/p21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAF1/p21 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAF1 increased clone sizes and numbers. WAF1 sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis- induced wild-type p53 was not inhibited by overexpression of antisense WAF1/p21. In a cell clone derived from line VMCub1 by stable transfection with wild-type p53 under the control of a metallothionein promotor, p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.     

Functional magnetic resonance imaging of median nerve stimulation in rats at 2.0 T

A cDNA clone, called CLB1, was isolated from a cDNA library from tomato (Lycopersicon esculentum) and characterized. The CLB1 cDNA contains an open reading frame of 1518 bp, and encodes a putative protein of 506 amino acids with a predicted molecular mass of 54,633 Da. The deduced CLB1 amino acid sequence contains a domain that exhibits from 26% to 37% identity with the Ca2+-dependent lipid-binding domains of cytosolic phospholipase A2, protein kinase C, Rabphilin-3A, and Synaptotagmin 1 of animals. Southern blot analysis indicates that the CLB1 gene belongs to a small gene family in the tomato genome. The CLB1 mRNA is preferentially expressed in fruit tissues.     

Effect of demographic factors, urinary peak flow rates, and Boyarsky symptom scores on patient treatment choice in benign prostatic hyperplasia

Thiopurine methyltransferase (TPMT) is a genetically polymorphic enzyme that catalyzes the S-methylation of thiopurine drugs such as 6-mercaptopurine. This genetic polymorphism is an important factor responsible for individual variation in thiopurine drug response. A cDNA for TPMT has been cloned from T84 human colon carcinoma cells. Northern blot analysis of multiple human tissues was performed with the T84 human colon carcinoma TPMT cDNA open reading frame (ORF) as a probe as one step toward understanding the molecular basis for the TPMT genetic polymorphism. Three mRNA species (approximately 1.4, 2.0, and 3.6 kb in length) were present in all tissues studied, including liver. However, none of these mRNAs matched the length of the 2.7 kb T84 TPMT cDNA. Therefore, it was important to clone a TPMT cDNA from a human drug-metabolizing organ such as the liver to determine whether its sequence matched that of the cDNA cloned from the T84 cell line. A human liver cDNA library was screened with the T84 TPMT cDNA ORF as a probe, and a 1.8 kb cDNA was isolated with a coding region sequence identical to that of the T84 TPMT cDNA. The TPMT cDNA ORF was then used to screen a human lymphocytes genomic DNA library in an attempt to clone the TPMT gene(s) in humans. Three intronless clones were isolated with identical ORF sequences that were 96% identical to that of the TPMT cDNA, but which contained multiple nucleotide substitutions and one deletion. The 3'- and 5'-flanking regions of one of the genomic DNA clones were sequenced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and identification of DNA fragments frequently deleted from tumor cell lines of human esophageal cancer

OBJECTIVE: To clone tumor suppressor genes associated with human esophageal cancer (EC). METHODS: DNA fragments deleted from cell lines of esophageal cancer EC8712 and EC8733 were isolated by using modified genomic substractive hybridization. Twenty EC tissues paired with adjacent normal mucosa were analysed by Southern blot and PCR with these fragments as probes. RESULTS: Seven DNA fragments deleted from EC cell lines obtained were named as 12H1, 12H2, 33H3, 33H4, 12B1, 33B2 and 33B3. Sequencing revealed their size of 512bp, 428bp, 509bp, 355bp, 680bp (partially sequencing), 519bp and 425bp, respectively. 12H1 and 33H3 showed high homology (98%) to a new repeated sequence in human genome. 33H4 displayed 87% nucleotide identity with human alphoid-like repetitive sequence located on chromosome 17. 12B1 was homologous (90%) to LINE 1 transoposon containing two open reading frames. 12H2, 33B2 and 33B3 were novel sequences. These DNA fragments were deleted in 20%-60% of EC tissues and in 10%-20% of adjacent mucosa. CONCLUSIONS: Loss of 33H4 may be accompanied by deletion of an adjacent gene. 12B1 as transposon may suppress the tumorigenesis by activating suppressor genes or/and by inactivating oncogenes. 12H1/33H3, 12H2, 33B2 and 33B3 may be candidates of tumor suppressor genes associated with the development and the progression of esophageal cancer.     

Prospective study of fluoxetine treatment and suicidal behavior in affectively ill subjects

A complementary DNA clone preferentially expressed in the gastrointestinal tract was obtained from a rat stomach library. The protein coded by the clone had a single carbohydrate recognition domain having conserved motifs for beta-galactoside binding and showed 67% amino acid identity with human galectin-2. The recombinant protein synthesized in Escherichia coli could bind to an asialofetuin column and was eluted with beta-galactopyranoside. From these observations, we named the protein rat galectin-2 coded by the cDNA. The rat galectin-2 was predominantly expressed in the epithelial cells of stomach. Thus this protein may form a mucin layer cross-linking with the beta-galactoside moiety of glycoproteins.