Inflammation-induced protein carbonylation contributes to poor prognosis for cholangiocarcinoma

Carbonylation is an irreversible and irreparable protein modification induced by oxidative stress. Cholangiocarcinoma (CCA) is associated with chronic inflammation caused by liver fluke infection. To investigate the relationship between protein carbonylation and CCA progression, carbonylated proteins were detected by 2D OxyBlot and identified by MALDI-TOF/TOF analyses in pooled CCA tissues in comparison to adjacent nontumor tissues and normal liver tissues. We identified 14 highly carbonylated proteins in CCA tissues. Immunoprecipitation and Western blot analyses of individual samples confirmed significantly greater carbonylation of serotransferrin, heat shock protein 70-kDa protein 1 (HSP70.1), and α1-antitrypsin (A1AT) in tumor tissues compared to normal tissues. The oxidative modification of these proteins was significantly associated with poor prognoses as determined by the Kaplan-Meier method. LC-MALDI-TOF/TOF mass spectrometry identified R50, K327, and P357 as carbonylated sites in serotransferrin, HSP70.1, and A1AT, respectively. Moreover, iron accumulation was significantly higher in CCA tissues with, compared to those without, carbonylated serotransferrin. We conclude that carbonylated serotransferrin-associated iron accumulation may induce oxidative stress via the Fenton reaction, and the carbonylation of HSP70.1 with antioxidative property and A1AT with protease inhibitory capacity may cause them to become dysfunctional, leading to CCA progression.     

The SNPs in the ACACA gene are effective on fatty acid composition in holstein milk

Fatty acid composition is an important economic trait for both dairy and beef cattle and controlled by genetic factors. Candidate genes controlling fatty acid composition may be found in fat synthesis and metabolism pathways. Acetyl-CoA carboxylase is the flux-determining enzyme in the regulation of fatty acid synthesis in animal tissues. One of two isozymes of this enzyme, acetyl-CoA carboxylase-α (ACACA), catalyses the first committed step of fatty acid synthesis in mammalian cytosol, leading to the biosynthesis of long-chain fatty acids. In the current study, the sequence comparison of the coding sequence (CDS) and two promoter regions (PIA and PIII) in bovine ACACA gene was performed between Japanese Black and Holstein cattle to detect nucleotide polymorphisms influencing fatty acid composition in milk and beef. Five single nucleotide polymorphisms (SNPs) were identified in the CDS region, 28 SNPs in the PIA region and three SNPs in the PIII region. Association study revealed that CCT/CCT type of PIII_#1, #2/PIA_#26 indicated a higher percentage of C14:0 in the milk of the Holstein cattle than CCT/GTC type (p = 0.050) and that a difference of the percentage of C16:0 was observed between CCT/CCT and GTC/GTC type (p = 0.023). CDS_#2 T/T type indicated a higher percentage of C18:0 than T/C type (p = 0.008). In addition, the Japanese Black cattle with CC/GT type of PIII_#1, #2 showed a higher percentage of C18:2 in the meat than those with GT/GT type (p = 0.025). Since PIII is the promoter specific to mammary gland during lactation, the altered expression of the ACACA gene owing to the SNPs in the PIII region may influence the fatty acid composition in the milk.     

Cloning and Sequencing of Two New Verotoxin 2 Variant Genes of Escherichia coli Isolated from Cases of Human and Bovine Diarrhea

We cloned and sequenced two new Verotoxin 2 (VT2) variant genes: one from an Escherichia coli strain from a case of bovine diarrhea and the other from an E. coli strain from a patient with diarrhea. The nucleotide and amino acid sequences of these two genes were highly homologous with, but distinct from those of the VT2, VT2vha, VT2vhb, SLT-IIv (VT2vp1) and SLT-IIva (VT2vp2) genes. Their nucleotide sequences were much more closely homologous to that of VT2vh than to that of VT2vp. Search for these two new genes in other Verocytotoxin-producing E. coli strains resulted in the isolation of 2 strains carrying one of the new VT2 variant genes, one strain from Tokyo and the other from Canada.     

Suicidal Feelings Interferes with Help-Seeking in Bullied Adolescents

Purpose

Being bullied is associated with the manifestation of suicidal feelings, which sharply increase in middle(-late) adolescence. Whether or not bullied middle(-late) adolescents with suicidal feelings seek help is therefore a critical issue, given that help-seeking plays a key role in the prevention of suicide. The aim of the present study is to investigate the effects of bullying, suicidal feelings and the interaction between these two factors on help-seeking behavior in adolescents.

Methods

Japanese middle(-late) adolescents (aged 15–18 years; n = 9484) were studied using self-report questionnaires. The rate of adolescents who actually sought help was examined for bullying status and suicidal feelings.

Results

The rate of adolescents who sought help was significantly higher when they were bullied (p<0.001) and also when they had mild suicidal feelings (p<0.001), but not when they displayed serious suicidal feelings. In the case of adolescents who were bullied, however, having suicidal feelings significantly decreased the rate of help-seeking (OR = 0.47, p<0.05 and OR = 0.32, p = 0.002 for having mild and serious suicidal feelings, respectively). The decrease was remarkable when suicidal feelings were serious. Specifically, the decrease was significant in seeking help from peers and family members, who are the most frequent source of the help for adolescents, when they had serious suicidal feelings (OR = 0.21, p<0.01 and OR = 0.13, p<0.001, respectively).

Conclusions

Suicidal feelings may interfere with help-seeking behavior, which could be critical in suicide prevention in bullied middle(-late) adolescents.     

Urinary Neutrophil Gelatinase-Associated Lipocalin: A Useful Biomarker for Tacrolimus-Induced Acute Kidney Injury in Liver Transplant Patients

Tacrolimus is widely used as an immunosuppressant in liver transplantation, and tacrolimus-induced acute kidney injury (AKI) is a serious complication of liver transplantation. For early detection of AKI, various urinary biomarkers such as monocyte chemotactic protein-1, liver-type fatty acid-binding protein, interleukin-18, osteopontin, cystatin C, clusterin and neutrophil gelatinase-associated lipocalin (NGAL) have been identified. Here, we attempt to identify urinary biomarkers for the early detection of tacrolimus-induced AKI in liver transplant patients. Urine samples were collected from 31 patients after living-donor liver transplantation (LDLT). Twenty recipients developed tacrolimus-induced AKI. After the initiation of tacrolimus therapy, urine samples were collected on postoperative days 7, 14, and 21. In patients who experienced AKI during postoperative day 21, additional spot urine samples were collected on postoperative days 28, 35, 42, 49, and 58. The 8 healthy volunteers, whose renal and liver functions were normal, were asked to collect their blood and spot urine samples. The urinary levels of NGAL, monocyte chemotactic protein-1 and liver-type fatty acid-binding protein were significantly higher in patients with AKI than in those without, while those of interleukin-18, osteopontin, cystatin C and clusterin did not differ between the 2 groups. The area under the receiver operating characteristics curve of urinary NGAL was 0.876 (95% confidence interval, 0.800–0.951; P<0.0001), which was better than those of the other six urinary biomarkers. In addition, the urinary levels of NGAL at postoperative day 1 (p = 0.0446) and day 7 (p = 0.0006) can be a good predictive marker for tacrolimus-induced AKI within next 6 days, respectively. In conclusion, urinary NGAL is a sensitive biomarker for tacrolimus-induced AKI, and may help predict renal event caused by tacrolimus therapy in liver transplant patients.     

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose.     

Copper-dependent DNA damage induced by hydrazobenzene,an azobenzene metabolite

Hydrazobenzene is carcinogenic to rats and mice and azobenzene is carcinogenic to rats. Hydrazobenzene is a metabolic intermediate of azobenzene. To clarify the mechanism of carcinogenesis by azobenzene and hydrazobenzene, we investigated DNA damage induced by hydrazobenzene, using ³²P-5′-end-labeled DNA fragments obtained from the c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Hydrazobenzene caused DNA damage in the presence of Cu(II). Piperidine treatment enhanced the DNA damage greatly, suggesting that hydrazobenzene caused base modification and liberation. However, azobenzene did not cause DNA damage even in the presence of Cu(II). Hydrazobenzene plus Cu(II) caused DNA damage frequently at thymine residues. Catalase and a Cu(I)-specific chelator inhibited Cu(II)-mediated DNA damage by hydrazobenzene. Typical ^·OH scavengers did not inhibit the DNA damage. The main active species is probably a metal oxygen complex, such as Cu(I)-OOH. Formation of 8-oxo-7, 8-dihydro-2′-deoxyguanosine was increased by hydrazobenzene in the presence of Cu(II). Oxygen consumption and UV-Visible spectroscopic measurements have shown that hydrazobenzene is autoxidized to azobenzene with H₂O₂ formation. It is considered that the metal-mediated DNA damage by hydrazobenzene through H₂O₂ generation may be relevant for the expression of carcinogenicity of azobenzene and hydrazobenzene.     

Function of a Glutamine Synthetase-Like Protein in Bacterial Aniline Oxidation via γ-Glutamylanilide

Acinetobacter sp. strain YAA has five genes (atdA1 to atdA5) involved in aniline oxidation as a part of the aniline degradation gene cluster. From sequence analysis, the five genes were expected to encode a glutamine synthetase (GS)-like protein (AtdA1), a glutamine amidotransferase-like protein (AtdA2), and an aromatic compound dioxygenase (AtdA3, AtdA4, and AtdA5) (M. Takeo, T. Fujii, and Y. Maeda, J. Ferment. Bioeng. 85:17-24, 1998). A recombinant Pseudomonas strain harboring these five genes quantitatively converted aniline into catechol, demonstrating that catechol is the major oxidation product from aniline. To elucidate the function of the GS-like protein AtdA1 in aniline oxidation, we purified it from recombinant Escherichia coli harboring atdA1. The purified AtdA1 protein produced gamma-glutamylanilide (γ-GA) quantitatively from aniline and l-glutamate in the presence of ATP and MgCl₂. This reaction was identical to glutamine synthesis by GS, except for the use of aniline instead of ammonia as the substrate. Recombinant Pseudomonas strains harboring the dioxygenase genes (atdA3 to atdA5) were unable to degrade aniline but converted γ-GA into catechol, indicating that γ-GA is an intermediate to catechol and a direct substrate for the dioxygenase. Unexpectedly, a recombinant Pseudomonas strain harboring only atdA2 hydrolyzed γ-GA into aniline, reversing the γ-GA formation by AtdA1. Deletion of atdA2 from atdA1 to atdA5 caused γ-GA accumulation from aniline in recombinant Pseudomonas cells and inhibited the growth of a recombinant Acinetobacter strain on aniline, suggesting that AtdA2 prevents γ-GA accumulation that is harmful to the host cell.     

The APC/C activator Cdh1 regulates the G2/M transition during differentiation of placental trophoblast stem cells

Differentiation of placental trophoblast stem (TS) cells to trophoblast giant (TG) cells is accompanied by transition from a mitotic cell cycle to an endocycle. Here, we report that Cdh1, a regulator of the anaphase-promoting complex/cyclosome (APC/C), negatively regulates mitotic entry upon the mitotic/endocycle transition. TS cells derived from homozygous Cdh1 gene-trapped (Cdh1^GT/GT) murine embryos accumulated mitotic cyclins and precociously entered mitosis after induction of TS cell differentiation, indicating that Cdh1 is required for the switch from mitosis to the endocycle. Furthermore, the Cdh1^GT/GT TS cells and placenta showed aberrant expression of placental differentiation markers. These data highlight an important role of Cdh1 in the G2/M transition during placental differentiation.