Nucleotide sequence of the 5'-terminal region of rat 18S ribosomal DNA

The 5'-terminal 597 base-pairs (bp) of the Sprague-Dawley rat 18S ribosomal RNA gee and 10 bp of the adjoining transcribed spacer have been sequenced. Previously sequenced 10 large oligonucleotides of rat 18S RNA were located in this region. This mammalian sequence has been compared with the known sequences of yeast and frog 18S rDNA's. The analysis indicates that 534 bp of the 597 bp (89%) are conserved between rat and frog sequences but only 75% of the nucleotides are conserved between rat and yeast in this region. Two large and two small sections have been identified where insertions have been introduced during evolution. Of these 58 bp long inserted sections of the rat rDNA sequence, 50 bp (86%) were G-C base-pairs.     

Topological considerations in protein structure. I. Disulfide linkage

The patterns of disulfide bridges in proteins were considered by using the concept of topological information content. It was proposed that the difference between topological informal ion content in the half-cystine residues of the native state and of the fully reduced form of a protein is related to the problems of the renaturation of the fully reduced and denatured protein. Specifically, when there is no difference between the topological information contents of the two states, the reduced protein is able to recover its native conformation. The concept reported in the present paper is consistent without exception with reported experimental results.     

Correlative Fine Structural,Behavioral, and Histochemical Analysis of Ascidian Blood Cells

Abstract The blood cells of a solitary ascidian, Halocynthia roretzi, were examined by electron microscopy (EM) with reference to their appearance by light microscopy (LM). In addition, their movement and stainability by vital dyes was observed by phase-contrast microscopy, and their stainability by Giemsa was also examined. Nine cell types were recognized: vacuolated cells, hyaline amoebocytes, small amoebocytes, granular amoebocytes, macrogranular cells, globular cells, lymphocyte-like cells, large basophilic cells and large granular cells. Vacuolated cells were found to possess various numbers of vacuoles containing strongly electron-dense materials and could be divided into at least three subgroups. Granular amoebocytes contained microfilaments and many granules of uniform size. Hyaline amoebocytes and small amoebocytes seemed to be specialized as phagocytes. Macrogranular cells and globular cells were not well characterized. In the blood of adult individuals, hemoblasts were rarely found, although lymphocyte-like cells were present. Each of two large cells, large basophilic cells and large granular cells, possessed novel granules or vacuoles, whose functions remain to be elucidated. The possible functions and relationships of these cells among various ascidian species are discussed.     

A pathway through interferon-gamma is the main pathway for induction of nitric oxide upon stimulation with bacterial lipopolysaccharide in mouse peritoneal cells.

Production of nitric oxide (NO) in response to bacterial lipopolysaccharide (LPS) was investigated using cultures of mouse peritoneal exudate cells (PEC) and the macrophage cell line RAW264.7. In the presence of anti-(interferon-gamma) (IFN-gamma), NO production was markedly suppressed in the PEC culture but not in the RAW264.7 culture. In the PEC culture, LPS induced both IFN-gamma production and activation of IFN response factor-1, which leads to the gene expression of inducible NO synthase, but neither was induced in the culture of RAW264.7 cells. In addition to anti-(IFN-gamma), antibodies against interleukin (IL)-12 and IL-18 showed a suppressive effect on LPS-induced NO production in the PEC culture, and these antibodies in synergy showed strong suppression. Stimulation of the PEC culture with IL-12 or IL-18 induced production of IFN-gamma and NO, and these cytokines, in combination, exhibited marked synergism. Stimulation of the culture with IFN-gamma induced production of NO, but not IL-12. The macrophage population in the PEC, prepared as adherent cells, responded well to LPS for IL-12 production, but weakly for production of IFN-gamma and NO. The macrophages also responded well to IFN-gamma for NO production. For production of IFN-gamma by stimulation with LPS or IL-12 + IL-18, nonadherent cells were required in the PEC culture. Considering these results overall, the indirect pathway, through the production of intermediates (such as IFN-gamma-inducing cytokines and IFN-gamma) by the cooperation of macrophages with nonadherent cells, was revealed to play the main role in the LPS-induced NO production pathway, as opposed to the direct pathway requiring only a macrophage population.     

Genetic Variation in VP7 Gene of Human Rotavirus Serotype 2 (G2 Type) Isolated in Japan,China, and Pakistan

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on sixteen human isolates of serotype 2 of rotavirus in Japan, China, and Pakistan and their genetic variations were examined. Comparative studies of their nucleotide and deduced amino acid sequences between the sixteen isolates and the HU5 strain revealed an overall homology of more than 94%. A higher degree of homology in nucleotides was observed among the sixteen isolates than between HU5 and the isolates. A total of thirteen amino acid residues frequently converted to another amino acid. Out of the thirteen, five amino acid residues belonging to the major neutralizing epitope regions (C, E, and F in this communication) converted frequently. From the amino acid sequences three subtypes, subtype 1, subtype 2, and intermediate, were suggested to be classified as previously reported for serotype 1 (Xin et al, Virology, 1993, 197: 813-816).     

Sequence analysis of T1 ribonuclease fragments of 18S ribosomal RNA by 5''-terminal labeling, partial digestion, and homochromatography fingerprinting.

The method employed to determine the sequence of a T1 RNase fragment, A-A-A-A-A-U-A-A-C-A-A-U-A-C-A-Gp, from Novikoff rat hepatoma 18S ribosomal RNA is described. This method is applicable to any oligoribonucleotide produced by specific endonucleases that leave the newly cleaved 5'-end free for labeling with polynucleotide kinase and gamma-(32p)-ATP. The (32p)-labeled oligoribonucleotide is subjected to partial endonucleolytic digestion and fractionated by two-dimensional homochromatography fingerprinting. The nucleotide sequence is determined by following mobility shifts of the labeled and partially digested oligoribonucleotides in homochromatography fingerprinting.     

Purification and characterization of Escherichia coli heat-stable enterotoxin II.

Escherichia coli heat-stable enterotoxin II (STII) was purified to homogeneity by successive column chromatographies from the culture supernatant of a strain harboring the plasmid encoding the STII gene. The purified STII evoked a secretory response in the suckling mouse assay and ligated rat intestinal loop assay in the presence of protease inhibitor, but the response was not observed in the absence of the inhibitor. Analyses of the peptide by the Edman degradation method and fast atom bombardment mass spectrometry revealed that purified STII is composed of 48 amino acid residues and that its amino acid sequence was identical to the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence (C. H. Lee, S. L. Mosely, H. W. Moon, S. C. Whipp, C. L. Gyles, and M. So, Infect. Immun. 42:264-268, 1983). STII has four cysteine residues which form two intramolecular disulfide bonds. Two disulfide bonds were determined to be formed between Cys-10-Cys-48 and Cys-21-Cys-36 by analyzing tryptic hydrolysates of STII.     

Regional and Temporal Alterations in Ca2+/Calmodulin-Dependent Protein Kinase II and Calcineurin in the Hippocampus of Rat Brain After Transient Forebrain Ischemia

We have investigated regional and temporal alterations in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and calcineurin (Ca2+/calmodulin-dependent protein phosphatase) after transient forebrain ischemia. Immunoreactivity and enzyme activity of CaM kinase II decreased in regions CA1 and CA3, and in the dentate gyrus, of the hippocampus early (6-12 h) after ischemia, but the decrease in immunoreactivity gradually recovered over time, except in the CA1 region. Furthermore, the increase in Ca2+/calmodulin-independent activity was detected up to 3 days after ischemia in all regions tested, suggesting that the concentration of intracellular Ca2+ increased. In contrast to CaM kinase II, as immunohistochemistry and regional immunoblot analysis revealed, calcineurin was preserved in the CA1 region until 1.5 days and then lost with the increase in morphological degeneration of neurons. Immunoblot analysis confirmed the findings of the immunohistochemistry. These results suggest that there is a difference between CaM kinase II and calcineurin in regional and temporal loss after ischemia and that imbalance of Ca2+/calmodulin-dependent protein phosphorylation-dephosphorylation may occur.     

Identification of miR-30e* Regulation of Bmi1 Expression Mediated by Tumor-Associated Macrophages in Gastrointestinal Cancer

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.