BACKGROUND: Cytological examination of intraoperative pleural or peritoneal lavage specimens is useful for predicting outcomes for patients with various carcinomas. There have been few reports regarding cytological examination of pleural lavage fluid in esophageal carcinoma. METHODS: Intraoperative pleural lavage fluid was collected before and after esophagectomy and was examined by Papanicolaou and Giemsa staining for 78 patients with esophageal carcinoma. RESULTS: Although epithelial cells were found for 29 patients, only blood cells were detected for 48. The remaining one patient exhibited no cells in the specimen. For 4 of 78 (5.2%) patients, tumor cells were detected in the pleural lavage fluid after esophagectomy. Three of these four patients had T4 tumors. CONCLUSIONS: Positive cytological findings for pleural lavage fluid, using Papanicolaou and Giemsa staining, is correlated with regrowth of residual tumor and poor prognosis in esophageal carcinoma.

Bronchoalveolar Lavage Fluid↗

PubMedDate not supplied

Differential expression of protein kinase C isozymes and small GTP-binding proteins during HL60 cell differentiation by retinoic acid and cyclic AMP: relation with phospholipase D (PLD) activation.

The differential expression of protein kinase C (PKC) isozymes and small GTP-binding proteins, and their relation to O2 generation and phospholipase D (PLD) activation were analyzed during the differentiation of human promyelocytic HL60 cells to neutrophil-like cells induced by either retinoic acid (RA) or dibutyryl cyclic AMP (dbcAMP). In response to either one of the inducers, nitroblue tetrazolium (NBT) reduction activity time-dependently increased. Although PLD activity was upregulated by dbcAMP-treatment, only a slight increase was observed in RA-treated cells. Small GTP-binding proteins Rac1, Rap1, and RhoA, which are reported to be implicated in O2- generation or PLD activation, were already expressed in undifferentiated HL60 cells and their significant changes were not detected during differentiation. The mRNAs of the cytosolic components of NADPH oxidase system, p47phox and p67phox, were present in trace amounts in undifferentiated cells. However, they rapidly increased in response to RA or dbcAMP. In response to either RA or dbcAMP, the increases were observed in cPKC isozymes (alpha, beta I, beta II) but not in other subtypes (delta, epsilon, theta, zeta) by both RT-PCR and Western blot analyses. In dbcAMP-treated cells PKC alpha increased remarkably, whereas PKC beta I and beta II mainly elevated in RA-treated cells. These results suggest the possibility that cPKCs are closely related to cell differentiation and that PKC alpha is involved in PLD activation.

Bucladesine↗

PubMedDate not supplied

Molecular cloning of a gene encoding translation initiation factor (TIF) from Candida albicans.

The differential display technique was applied to compare mRNAs from two clinical isolates of Candida albicans with different virulence; high (potent strain, 16240) and low (weak strain, 18084) extracellular phospholipase activities. Complementary DNA fragments corresponding to several apparently differentially expressed mRNAs were recovered and sequenced. A complementary DNA fragment seen distinctly in the potent phospholipase producing strain was highly homologous to the yeast translation initiation factor (TIF). The selected DNA fragment was then used as a probe to isolate its corresponding complementary DNA clone from a library of C. albicans genomic DNA. The sequence of isolated gene revealed an open reading frame of 1194 nucleotides with the potential to encode a protein of 397 amino acids with a predicted molecular weight of 43 kDa. Over its entire length, the amino acid sequence showed strong homology (78-89%) to Saccharomyces cerevisiae TIF and (63-80%) to mouse eIF-4A proteins. Therefore, our C. albicans gene was identified to be TIF (Ca TIF). Northern blot analysis in the two strains of C. albicans revealed that Ca TIF expression is 1.5-fold higher in the potent phospholipase producing strain. The restriction endonuclease digestion of genomic DNA from this potent strain revealed at least two hybridized bands in Southern blot analysis, suggesting two or more closely related sequences in the C. albicans genome.

Amino Acid Sequence↗

PubMedDate not supplied

Molecular cloning of a gene encoding acid alpha-glucosidase from Tetrahymena pyriformis.

Lysosomal acid alpha-glucosidase is essential for the degradation of glycogen to glucose in lysosomes. The ciliated protozoan Tetrahymena pyriformis secretes acid alpha-glucosidase into its culture medium. We have earlier reported the purification and characterization of acid alpha-glucosidase from T. pyriformis. The exact molecular mechanism of secretion of this enzyme has not yet been clarified. In the present study we have isolated a full length cDNA clone encoding acid alpha-glucosidase from T. pyriformis. The isolated clone (3019 bp) contained an open reading frame encoding 923 amino acids, and has an estimated molecular mass of 104 kDa. Northern blot analysis revealed that the isolated cDNA hybridized to a 2.8-kb mRNA transcript. N-terminal amino acids after the first methionine fulfilled the requirement of a signal peptide. The deduced amino acid sequence contains the amino acid sequences determined of several peptides derived from the purified enzyme, and was found to have 34% identity and 45% similarity with that of human lysosomal enzyme, with 75% identity in the 16 amino acids at the proposed active site.

Amino Acid Sequence↗

PubMedDate not supplied

Cloning and sequencing of a cDNA encoding glyceraldehyde-3-phosphate dehydrogenase from Tetrahymena thermophila: growth-associated changes in its mRNA expression.

Glyceraldehyde-3-phosphate dehydrogenase is a key enzyme in the glycolytic pathway. Since its transcript levels do not vary in most experimental conditions, it has been often used as a control in northern blot or reverse transcriptase-polymerase chain reaction analysis. We have cloned and sequenced a gene encoding glyceraldehyde-3-phosphate dehydrogenase (Tthgapdh) from Tetrahymena thermophila cDNA library and determined whether the Tthgapdh mRNA is a loading control in gene expression studies of T. thermophila cell. The open reading frame encoded a protein of 341 amino acid residues (36.8 kDa) containing a nicotinamide adenine dinucleotide-binding domain and a catalytic domain, which was highly similar to those of other organisms. Its mRNA levels at different growth stages were examined by northern blot analysis. The fragment of the isolated cDNA was hybridized to a 1.3-kb mRNA transcript. There was a marked increase in Tthgapdh mRNA level at the mid-exponential phase, followed by a gradual decrease. Therefore, much caution should be made to use Tthgapdh mRNA as an internal standard for northern blot analysis in Tetrahymena.

Amino Acid Sequence↗

PubMedDate not supplied

Enzymatic characterization of phospholipase D of protozoan Tetrahymena cells.

Phospholipase D (PLD), which is present in plant, bacterial, and mammalian cells, has been proposed to be involved in a number of cellular processes including transmembrane signaling and membrane deterioration. We demonstrated the existence of evolutionally related PLD activity in the unicellular eukaryotic protozoan Tetrahymena. The partial characterization of this enzyme showed that PLD in Tetrahymena cells was a neutral phospholipase, which catalyzed both transphosphatidylation and hydrolysis reac tions. The activity was markedly stimulated by phosphatidylinositol 4, 5-bisphosphate (PIP2) but was insensitive to phorbol 12-myristate 13-acetate (PMA) and guanosine 5'-3-O-(thio)triphosphate (GTPgammaS), suggesting that it is a PIP2-dependent PLD and that protein kinase C (PKC) and GTP-binding proteins are not implicated in the regulation of this enzyme. For its maximal activity Ca2+ was not required. This enzyme was also capable of hydrolyzing phosphatidylcholine (PC) but not phosphatidylethanolamine (PE), implying that PC was a preferred substrate. Subcellular fractionation showed that PLD-like activity localized mainly to the membrane fraction, especially microsomes. As an initial step to explore the functions of PLD in Tetrahymena, the PLD-like activity was determined during the different culture phases, and it was found to be significantly and transiently elevated in the early logarithmic phase, indicating its possible role in the development of Tetrahymena.

1-Butanol↗

PubMedDate not supplied

Analysis of macroscopic appearance and p53 expression in superficial esophageal carcinoma.

BACKGROUND/AIMS: The purpose of the present study was to investigate the relationship between macroscopic appearance of superficial esophageal carcinoma, with particular attention to the horizontal and vertical extent of tumor growth, clinicopathologic findings and p53 expression. METHODOLOGY: Eighty-seven patients with superficial esophageal carcinoma were divided into three groups: 1) group A, patients with protruding or distinct depressed lesions (n = 28); 2) group B, patients with superficial and flat lesions > or = 5 cm in length (n = 45); and, group C, patients with superficial and flat lesions (5 cm in length (n = 14). Tumors were examined immunohistochemically for p53 expression. RESULTS: The incidence of submucosal invasion, lymph node metastasis and lymphatic invasion was significantly higher in group A than in groups B and C. The rate of p53 expression was significantly lower in group B than in the other two groups. The prognosis in groups B and C was better than that in group A. CONCLUSIONS: Vertical extent was more strongly associated with tumor depth, lymph node metastasis and prognosis than was horizontal extent, although p53 overexpression was related to both the vertical and horizontal extent of tumors. Analysis of the macroscopic appearance of superficial esophageal carcinoma is useful in choosing treatment strategies.

Aged↗

PubMedDate not supplied

Expression of p53 and p21 is useful for the prediction of preoperative chemotherapeutic effects in esophageal carcinoma.

The p53 and p21 genes are associated with G1 arrest during the cell cycle and with apoptosis, both of which have a close relationship with the effect of chemotherapy. In this study, we investigated the correlation between p53 and p21 expression in biopsy specimens and the histological effect of chemotherapy in esophageal carcinoma. A total of 30 patients with esophageal squamous cell carcinoma received preoperative chemotherapy, then underwent esophagectomy with lymph node dissection. The response rate of primary lesion and metastatic nodes was 20.0% and 25.9%, respectively. The positive rate of p53 and p21 expression was 56.7% and 36.7%, respectively. Preoperative chemotherapy against primary lesions was ineffective in all the patients who expressed p53, but not p21. In contrast, chemotherapy was effective against metastatic lymph nodes which were p53 negative but p21 positive. These findings suggest that p21 positive expression in the absence of p53 is associated with favorable effects of preoperative chemotherapy in patients with esophageal carcinoma. Therefore, the expression of these genes should be examined in biopsy specimens to predict the chemotherapeutic outcomes in patients with esophageal carcinoma.

Adult↗

PubMedDate not supplied

Molecular cloning of a Rho family, CDC42Ca gene from Candida albicans and its mRNA expression changes during morphogenesis.

The small GTP-binding protein family regulates various cell functions in mammalian and yeast cells. In the yeast Saccharomyces cerevisiae it has been known to be involved in vegetative growth. As an initial attempt to explore the involvement of CDC42, a member of this family, in the regulation of morphological changes in Candida albicans, we isolated a gene encoding this protein (CDC42Ca) from this fungus. The sequence of isolated gene revealed an open reading frame of 570 nucleotides with the potential to encode a protein of 190 amino acids with a predicted molecular weight of 20.5 kDa. The deduced amino acid sequence was highly homologous to CDC42s from yeast (87.8%), human (76.4%) and Caenorhabditis elegans (73.7%). The CDC42Ca mRNA level showed a transient increase with a peak at 2 h after the fresh medium shift (28 degrees C) when cells synchronously formed buds, whereas it displayed a gradual increase up to 12 h after the medium shift (37 degrees C) with elongation of germ tubes. This suggests that CDC42 may play a role in the bud emergence and also germ tube formation in C. albicans.

Amino Acid Sequence↗