MiR-376c Down-Regulation Accelerates EGF-Dependent Migration by Targeting GRB2 in the HuCCT1 Human Intrahepatic Cholangiocarcinoma Cell Line

MicroRNA miR-376c was expressed in normal intrahepatic biliary epithelial cells (HIBEpiC), but was significantly suppressed in the HuCCT1 intrahepatic cholangiocarcinoma (ICC) cell line. The biological significance of the down-regulation of miR-376c in HuCCT1 cells is unknown. We hypothesized that miR-376c could function as a tumor suppressor in these cells. To test this hypothesis, we sought the targets of miR-376c, and characterized the effect of its down-regulation on HuCCT1 cells. We performed proteomic analysis of miR-376c-overexpressing HuCCT1 cells to identify candidate targets of miR-376c, and validated these targets by 3′-UTR reporter assay. Transwell migration assays were performed to study the migratory response of HuCCT1 cells to miR-376c overexpression. Furthermore, microarrays were used to identify the signaling that were potentially involved in the miR-376c-modulated migration of HuCCT1. Finally, we assessed epigenetic changes within the potential promoter region of the miR-376c gene in these cells. Proteomic analysis and subsequent validation assays showed that growth factor receptor-bound protein 2 (GRB2) was a direct target of miR-376c. The transwell migration assay revealed that miR-376c significantly reduced epidermal growth factor (EGF)-dependent cell migration in HuCCT1 cells. DNA microarray and subsequent pathway analysis showed that interleukin 1 beta and matrix metallopeptidase 9 were possible participants in EGF-dependent migration of HuCCT1 cells. Bisulfite sequencing showed higher methylation levels of CpG sites upstream of the miR-376c gene in HuCCT1 relative to HIBEpiC cells. Combined treatment with the DNA-demethylating agent 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor trichostatin A significantly upregulated the expression of miR-376c in HuCCT1 cells. We revealed that epigenetic repression of miR-376c accelerated EGF-dependent cell migration through its target GRB2 in HuCCT1 cells. These findings suggest that miR-376c functions as a tumor suppressor. Since metastasis is the major cause of death in ICC, microRNA manipulation could lead to the development of novel anti-cancer therapy strategies for ICC.     

Module-based construction of plasmids for chromosomal integration of the fission yeast Schizosaccharomyces pombe

Integration of an external gene into a fission yeast chromosome is useful to investigate the effect of the gene product. An easy way to knock-in a gene construct is use of an integration plasmid, which can be targeted and inserted to a chromosome through homologous recombination. Despite the advantage of integration, construction of integration plasmids is energy- and time-consuming, because there is no systematic library of integration plasmids with various promoters, fluorescent protein tags, terminators and selection markers; therefore, researchers are often forced to make appropriate ones through multiple rounds of cloning procedures. Here, we establish materials and methods to easily construct integration plasmids. We introduce a convenient cloning system based on Golden Gate DNA shuffling, which enables the connection of multiple DNA fragments at once: any kind of promoters and terminators, the gene of interest, in combination with any fluorescent protein tag genes and any selection markers. Each of those DNA fragments, called a ‘module’, can be tandemly ligated in the order we desire in a single reaction, which yields a circular plasmid in a one-step manner. The resulting plasmids can be integrated through standard methods for transformation. Thus, these materials and methods help easy construction of knock-in strains, and this will further increase the value of fission yeast as a model organism.     

Detection of Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, in dialysis fluid of patients with uremia

In order to estimate the exposure levels of mutagenic and carcinogenic heterocyclic amines in humans, we developed a high-performance liquid chromatography method to detect 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in dialysis fluid of patients with uremia. Using this methods, dialysis fluid of 12 patients who had received hemodialysis treatment or continuous ambulatory peritoneal dialysis was examined. Trp-P-1 was detected in dialysate of all uremic patients (727 +/- 282 pmoles, n = 12). In patients who had been treated with continuous ambulatory peritoneal dialysis, the average amount of Trp-P-1 found in whole dialysate (6 l) per day was 710 +/- 203 pmoles (mean +/- S.D., n = 8). Moreover, Trp-P-2 could be detected in 5 out of 12 patients (206 +/- 85 pmoles, n = 5). These results indicate that patients with uremia are actually exposed to carcinogenic tryptophan pyrolysis products. The average exposure level of Trp-P-1 in uremic patients apparently exceeded 710 pmoles (150 ng) per day.     

Low temperature spectrophotometry of the phototransformation of Pfr to Pr in pelletable pea phytochrome

Manabe, K. 1987. Low temperature spectrophotometry of the phototransformation of P_fr to P_r, in pelletable pea phytochrome.
Low temperature spectrophotometry was used to study the phototransformation of P_fr to P_r in 1000–7000 g pelletable fractions extracted from dark grown pea ( Pisum sativum L. cv. Alaska) epicotyls which had been irradiated with red and then far-red light. At -170°C, far-red irradiation of the pelletable phytochrome which had been pre-irradiated with saturating fluence of red light before freezing caused formation of an intermediate (named I₆₆₀), the difference spectrum of which showed a marked ab-sorbance decrease at 740 nm and a concomitant small increase at about 660 nm. The inermediate I₆₆₀ was converted to another intermediate (I₆₆₀) when it was warmed above -80°C. The difference spectrum of this intermediate showed a positive peak at 670 nm. This intermediate was photoconverted to P_fr by red irradiation and also underwent dark reversion to P_fr at -60°C. I₆₆₀ formed P_r if the temperature was above -10°C. The basic features of the phytochrome intermediates resemble those obtained in vivo and in degraded purified phytochrome.     

Purification and Characterization of a Substrate-Size-Recognizing Metalloendopeptidase from Streptococcus cremoris H61

During the ripening of Gouda-type cheese, two kinds of endopeptidases were found to participate in the degradation of αs1-CN(f1-23), a specific product from αs1-casein hydrolyzed by chymosin. One of the endopeptidases, lactic acid bacteria endopeptidase (LEP-II), which can recognize the size of its substrates, has already been purified and characterized (T. R. Yan, N. Azuma, S. Kaminogawa, and K. Yamauchi, Eur. J. Biochem. 163:259-265, 1987). The other endopeptidase, LEP-I, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme appeared to be monomeric, with an apparent molecular weight of 98,000, and its isoelectric point was 5.1. For the hydrolysis of αs1-CN(f1-23), the enzyme had an optimum pH and temperature of 7.0 to 7.5 and 40°C, respectively. Its activity was inhibited by such chelating agents as EDTA and 1,10-phenanthrolin, and it could be fully reactivated by Mn²⁺. Inhibitors specific for serine and thiol proteases had no effect on the protease activity. The enzyme showed a high affinity toward the Glu-Asn peptide bond of αs1-CN(f1-23) and αs1-CN(f91-100) but showed no hydrolysis activity toward αs1-CN(f1-52), αs1-CN(61-122), αs1-CN(136-196), αs1-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin. The K_m and V_max of LEP-I for αs1-CN(f1-23) were 14.2 pM and 139 U, respectively.     

Longitudinal changes of dominance rank among the females of the Koshima group of Japanese monkeys

The changes of dominance rank among female Japanese monkeys of the Koshima group over a period of 29 years from 1957 were studied. The dominance rank order was relatively stable in the early population growing phase, while large scale-changes of dominance rank order occurred successively in the phase of population decrease brought about by the severe control of artificial feeding after 1972. Nevertheless, the rank order of several females of the highest status was stable. Furthermore, the reproductive success of these highest status females was high (Mori, 1979a;Watanabe et al., in prep.). Divergence of the dominance rank order fromKawamura's rules (Kawamura, 1958) was observed in the following respects: (1) Some females significantly elevated their rank depending on the leader males. (2) If mothers died when their daughters were still juveniles or nulliparous, the dominance rank of some of these offspring females was significantly lower than the mother's one. However 55% of daughters which lost their mothers at a young age inherited the mother's rank. (3) Dominance among sisters whose mother had died when at least one of the daughters was under 6 years old followed the rule of youngest ascendancy in 60% (Kawamura, 1958), and in 80% when both of the daughters were nulliparous at the mother's death. The mean rate of aggressive interactions for each female with subordinates to her was calculated by dividing the total aggressive interactions between the female in question and her subordinates by the number of subordinate females to the female in question. A female which showed a high rate of aggressive interactions with her subordinates was categorized as an “Attacker”, and a female showing a lower rate was categorized as a “Non-attacker”. Similarly, categories of “Attacked”, and “Non-attacked” were distinguished by using the rate of aggressive interactions with dominant females. Several females which were once categorized in one category in a year were repeatedly categorized in the same category over different years. The “Attacked” tended to be females of higher rank, and “Non-attackers” tended to be females of lower rank. “The second-higher-status females”, were “Attacked”, and their rank was unstable. In particular, females of lower rank within the lineage of the highest rank suffered this kind of severe status. Most of the daughters of these females showed a sharp drop of rank, and died when they were still at a young age, i.e. “the second-higher-status females” displayed low fitness. “Non-attackers” were significantly “Non-attacked”; i.e. they were females which showed a non-social attitude. Females which underwent a drop of rank tended to be “Non-attackers”. The most important factor which determined the females' rank was the memory of their dominance relations under the influence of their mother [dependent rank (Kawai, 1958)] in their early life during development. This finding corresponds well with the results in baboons obtained byWalter (1980); the target females of aggressive interactions by adolescent females were determined by the rank of the mothers when these adolescent females were born.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Evidence that chromium is an essential factor for biological activity of low-molecular-weight, chromium-binding substance

Biologically active Low-molecular-weight, chromium-binding substance (LMCr) has been isolated from the liver of rabbits injected with potassium bicarbonate and cow's milk. This substance enhances glucose oxidation and lipogenesis from glucose in rat adipocytes [Yamamoto A. et al. (1987) Eur. J. Biochem. 165, 627-631; (1988) J. Nut. 118, 39-45]. LMCr was shown to lose its activity almost completely as an effectant of glucose metabolism when Cr was deleted under acidic conditions and separated from LMCr by EDTA-chelation and successive molecular-sieve chromatography. Reincorporation of Cr3+ into apo-LMCr resulted in 50-90% recovery of its original activity. These findings suggest that the biological activity of LMCr resides in Cr.     

Relationship between general or specific immunoreactivity and prognosis in postoperative patients with hepatocellular carcinoma

Summary The levels of a variety of immunological parameters were examined in 203 preoperative patients with hepatocellular carcinoma (HCC) at various stages (I–IV). The changes in the peripheral blood lymphocyte (PBL) count, the serum level of immunosuppressive acidic protein and the degree of the skin reaction to purified protein derivative were associated significantly with the stage of HCC progression. However, the percentages of lymphocyte subsets, mitogenic responsiveness of PBL and serum immunoglobulin concentration remained at the levels of stage I. Further study demonstrated that in patients undergoing hepatic artery ligation, there were statistically significant correlations between the PBL count, immunosuppressive acidic protein concentration and intensity of the skin reaction to purified protein derivative, assayed 1 month after surgery, and the prognosis. HCC-specific immunity was examined in 34 patients treated by hepatic resection or hepatic artery ligation using in vitro responses of PBL to HCC extracts (ATS test). This test was performed using culture medium containing added arginine. None of the PBL from the patients showed a positive response to allogeneic HCC extracts, but the PBL from 12 patients (9 hepatic resections, 3 hepatic artery ligations) were stimulated significantly (SI 2.5) with autologous HCC extracts. In 7 of 9 hepatic resection patients who were positive in the ATS test, tumor recurrence was identified. Statistical analysis indicated that the ATS test result was significantly correlated with tumor recurrence in hepatic resection patients. Autologous-PBL-stimulating activities were isolated in a fraction at pH 8.3 and in fractions at pH 6.7–7.0 by chromatofocusing of the crude extract. Although identification of the HCC-specific antigen remains to be done, use of the above fractions may simplify the ATS test procedure and improve its sensitivity.     

Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.