Real-time monitoring of liver damage during experimental ischaemia-reperfusion using a nitric oxide sensor.

BACKGROUND: Hepatic ischaemia-reperfusion (IR) injury may lead to liver damage during liver surgery, and intrahepatic nitric oxide (NO) levels may play a role in this context. The aim of this study was to demonstrate real-time changes in intrahepatic NO concentration during IR and to correlate potential hepatic NO production with liver damage using a selective NO sensor. METHODS: Wistar rats were exposed to 15 min of hepatic ischaemia followed by reperfusion, after which changes in intrahepatic NO levels were measured using an NO sensor. Additionally, rats were exposed to five successive periods of IR, each consisting of 15 min ischaemia followed by 5 or 15 min reperfusion, and hepatic damage was evaluated by blood tests and histological examination. Hepatic expression of Akt, phosphorylated Akt, endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS was examined at different time points during and after IR by western blot and immunohistochemical analysis. RESULTS: During ischaemia, intrahepatic NO levels increased and reached a plateau at approximately 10 min. Repeated 15 min ischaemia-5 min reperfusion cycles reduced the maximum amount of NO produced during ischaemia gradually, and almost no NO production was observed during the fifth period of ischaemia. NO production following repeated ischaemia was proportional to the degree of hepatic viability. Phosphorylated eNOS was upregulated and correlated with the level of NO production during hepatic ischaemia. CONCLUSION: Intrahepatic NO levels decrease during repeated IR in rats. Real-time monitoring of intrahepatic NO levels is useful for the prediction of IR-related liver injury during experimental liver surgery.

Multifunctional transcription factor TFII-I is an activator of BRCA1 function.

BACKGROUND: The TFII-I is a multifunctional transcriptional factor known to bind specifically to several DNA sequence elements and to mediate growth factor signalling. A microdeletion at the chromosomal location 7q11.23 encoding TFII-I and the related family of transcription factors may result in the onset of Williams-Beuren syndrome, an autosomal dominant genetic disorder characterised by a unique cognitive profile, diabetes, hypertension, anxiety, and craniofacial defects. Hereditary breast and ovarian cancer susceptibility gene product BRCA1 has been shown to serve as a positive regulator of SIRT1 expression by binding to the promoter region of SIRT1, but cross talk between BRCA1 and TFII-I has not been investigated to date. METHODS: A physical interaction between TFII-I and BRCA1 was explored. To determine pathophysiological function of TFII-I, its role as a transcriptional cofactor for BRCA1 was investigated. RESULTS: We found a physical interaction between the carboxyl terminus of TFII-I and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous TFII-I and BRCA1 form a complex in nuclei of intact cells and formation of irradiation-induced nuclear foci was observed. We also showed that the expression of TFII-I stimulates the transcriptional activation function of BRCT by a transient expression assay. The expression of TFII-I also enhanced the transcriptional activation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed the intrinsic mechanism that TFII-I may modulate the cellular functions of BRCA1, and provide important implications to understand the development of breast cancer.

Lecithin-cholesterol acyltransferase (LCAT) deficiency without mutations in the coding sequence: a case report and literature review.

Familial lecithin-cholesterol acyltransferase (LCAT) deficiency (FLD) is a rare genetic disease characterized by corneal opacities, normocytic anemia, dyslipidemia, and proteinuria progressing to chronic renal failure. In all FLD cases, a mutation has been found in the coding sequence of the LCAT gene. FLD is clinically distinguished from an acquired form of LCAT deficiency by the presence of corneal opacities. Here we describe a 36-year-old woman presenting with clinical, pathological, and laboratory data compatible with FLD. Her mother and elder sister had corneal opacities. However, genetic analysis revealed there were no mutations in the LCAT coding sequences and no alterations in LCAT mRNA expression. Furthermore, we were unable to find any underlying conditions that may lead to LCAT deficiency. The present case therefore demonstrates that LCAT deficiency may be caused by factors other than mutations in the coding sequence and we suggest that a translational or posttranslational mechanism may be involved.

Identification of DBC1 as a transcriptional repressor for BRCA1.

BACKGROUND: DBC1/KIAA1967 (deleted in breast cancer 1) is a putative tumour-suppressor gene cloned from a heterozygously deleted region in breast cancer specimens. Caspase-dependent processing of DBC1 promotes apoptosis, and depletion of endogenous DBC1 negatively regulates p53-dependent apoptosis through its specific inhibition of SIRT1. Hereditary breast and ovarian cancer susceptibility gene product BRCA1, by binding to the promoter region of SIRT1, is a positive regulator of SIRT1 expression. METHODS: A physical interaction between DBC1 and BRCA1 was investigated both in vivo and in vitro. To determine the pathophysiological significance of DBC1, its role as a transcriptional factor was studied. RESULTS: We found a physical interaction between the amino terminus of DBC1 and the carboxyl terminus of BRCA1, also known as the BRCT domain. Endogenous DBC1 and BRCA1 form a complex in the nucleus of intact cells, which is exported to the cytoplasm during ultraviolet-induced apoptosis. We also showed that the expression of DBC1 represses the transcriptional activation function of BRCT by a transient expression assay. The expression of DBC1 also inhibits the transactivation of the SIRT1 promoter mediated by full-length BRCA1. CONCLUSION: These results revealed that DBC1 may modulate the cellular functions of BRCA1 and have important implications in the understanding of carcinogenesis in breast tissue.

Kimura's disease: effects of age on clinical presentation.

BACKGROUND: Kimura's disease (KD) is known to be dominant among young Asian men, but it can also occur in middle- and advanced-aged people. The clinical characteristics of KD, especially by age, are not well known. AIM: This study was performed to investigate the effects of age on the clinical characteristics of KD. DESIGN: We conducted a case series study. METHODS: All case studies of patients diagnosed with KD were collected via a PubMed search of studies published until August 2018. The data were analyzed by age group. RESULTS: In total, 215 studies were reviewed (238 patients; mean age of 36 years). The male:female ratio was 4:1 overall, 17:1 in patients aged <20 years, 4:1 in patients aged 20-39 years and 2:1 in patients aged ≥40 years (P = 0.01). The percentage of patients with pruritus was 15.4% overall, 3.8% in patients aged <20 years, 15.5% in patients aged 20-39 years and 21.7% in patients aged ≥40 years (P = 0.02). The time to diagnosis was 5.3 years overall, 3.2 years in patients aged <20 years, 4.7 years in patients aged 20-39 years and 7.1 years in patients aged ≥40 years (P < 0.01). CONCLUSIONS: The proportion of female patients affected the incidence of pruritus, and the time to diagnosis increased as the patients' age increased. There were no significant age-related differences in region/race, complications, multiplicity, laterality, anatomical distribution, maximum size, eosinophil count, immunoglobulin E level, initial treatment, recurrence or outcomes. This may be useful information for the diagnosis of KD.

The oncogenic mutation in the pleckstrin homology domain of AKT1 in endometrial carcinomas.

BACKGROUND: The phosphatidylinositol 3'-kinase (PI3K)-AKT pathway is activated in many human cancers and plays a key role in cell proliferation and survival. A mutation (E17K) in the pleckstrin homology domain of the AKT1 results in constitutive AKT1 activation by means of localisation to the plasma membrane. The AKT1 (E17K) mutation has been reported in some tumour types (breast, colorectal, ovarian and lung cancers), and it is of interest which tumour types other than those possess the E17K mutation. METHODS: We analysed the presence of the AKT1 (E17K) mutation in 89 endometrial cancer tissue specimens and in 12 endometrial cancer cell lines by PCR and direct sequencing. RESULTS: We detected two AKT1 (E17K) mutations in the tissue samples (2 out of 89) and no mutations in the cell lines. These two AKT1 mutant tumours do not possess any mutations in PIK3CA, PTEN and K-Ras. INTERPRETATION: Our results and earlier reports suggest that AKT1 mutations might be mutually exclusive with other PI3K-AKT-activating alterations, although PIK3CA mutations frequently coexist with other alterations (such as HER2, K-Ras and PTEN) in several types of tumours.

Renal artery pseudoaneurysm occurring after laparoscopic partial nephrectomy.

Renal artery pseudoaneurysm (RAP) is rare, and has been reported after renal biopsy and percutaneous renal surgery. We report a case of RAP after laparoscopic partial nephrectomy for renal cell carcinoma.

Postoperative pain status after intraoperative systemic dexmedetomidine and epidural neostigmine in patients undergoing lower abdominal surgery.

BACKGROUND AND OBJECTIVES: To determine whether intraoperative systemic dexmedetomidine improves postoperative pain and interacts with epidural neostigmine to produce analgesic effects. METHODS: Sixty patients undergoing gynaecological surgery were randomly divided into four groups to receive epidural neostigmine and/or systemic dexmedetomidine: control (Group C), epidural neostigmine (Group N), systemic dexmedetomidine (Group D) and co-administered neostigmine and dexmedetomidine (Group ND). Epidural neostigmine (0.3 mg) was administered with 10 mL of 0.75% ropivacaine before the induction of general anaesthesia. Systemic dexmedetomidine (loading dose of 1 mug kg-1 over 10 min followed by 0.4 mug kg-1 h-1) was infused after the induction of general anaesthesia and continued until the end of surgery. The pain status of patients was assessed using the visual analogue scale at 2, 4, 6, 24 and 72 h postoperatively. RESULTS: Intraoperative systemic dexmedetomidine alone did not reduce postoperative pain scores. However, co-administered neostigmine and dexmedetomidine significantly decreased scores at 24 and 72 h (Group C: 3.0 [1.0-5.8] and 2.0 [0.3-3.0]; Group N: 1.5 [0.3-3.4] and 0 [0-1.3]; Group D: 3.5 [0-5.0] and 0 [0-1.4]; and Group ND: 0 [0-1.0]* and 0 [0-0]; median [interquartile range] *P = 0.0031, P = 0.0045 compared with Group C). CONCLUSIONS: The intraoperative systemic infusion of dexmedetomidine alone at doses causing sedation does not result in postoperative analgesic effects. However, the co-administration of systemic dexmedetomidine and epidural neostigmine at higher doses may be a useful method to improve postoperative pain although side-effects have to be evaluated.

Aqueous extract of Arbutus unedo inhibits STAT1 activation in human breast cancer cell line MDA-MB-231 and human fibroblasts through SHP2 activation.

Arbutus unedo L. has been for a long time employed in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes. Signal transducer and activator of transcription 1 (STAT1) is a fascinating and complex protein with multiple yet contrasting transcriptional functions. Although activation of this nuclear factor is finely regulated in order to control the entire inflammatory process, its hyper-activation or time-spatially erroneous activation may lead to exacerbation of inflammation. The modulation of this nuclear factor, therefore, has recently been considered as a new strategy in the treatment of inflammatory diseases. In this study, we present data showing that the aqueous extract of Arbutus unedo's leaves exerts inhibitory action on interferon-gamma (IFN-gamma) elicited activation of STAT1, both in human breast cancer cell line MDA-MB-231 and in human fibroblasts. This down-regulation of STAT1 is shown to result from a reduced tyrosine phosphorylation of STAT1 protein. Evidence is also presented indicating that the inhibitory effect of this extract may be mediated through enhancement of tyrosine phosphorylation of SHP2 tyrosine phosphatase. The modulation of this nuclear factor turns out into the regulation of the expression of a number of genes involved in the inflammatory response such as inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1). Taken together, our results suggest that the employment of the Arbutus unedo aqueous extract is promising, at least, as an auxiliary anti-inflammatory treatment of diseases in which STAT1 plays a critical role.

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages.

When cells of mouse myelomonocytic leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Iad, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.

Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility.

Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.

Cloning of a cDNA encoding an importin-alpha and down-regulation of the gene by light in rice leaves.

The import of nuclear proteins into nuclei begins with recognition of nuclear localization signal-harboring proteins and binding to a nuclear pore targeting complex. A cDNA for an importin-alpha protein, a subunit of the complex, was isolated from rice plants. The amino acid sequence deduced from the nucleotide sequence of the cDNA exhibited a high homology to those of importin-alpha proteins from many organisms such as Arabidopsis thaliana, Saccharomyces cerevisiae, human, mouse, Xenopus laevis and Drosophila melanogaster. Down-regulation of the transcription by light was shown in the leaves of light- and dark-grown seedlings by RNA blot analysis. The down-regulation was specific to leaves, whereas no light effect was observed in root tissues or calli, in which higher levels of the transcript were detected.

Construction of a novel suicide vector: selection for Escherichia coli HB101 recombinants carrying the DNA insert.

We constructed a new type of cloning vector, pERISH2, that transforms Escherichia coli HB101 only when a foreign DNA fragment is ligated into the cloning site of the plasmid vector. Plasmid pERISH2 carries the rcsB gene which is derived from the chromosome of E. coli HB101 and is involved in the regulation of colanic acid production. When E. coli HB101 is transformed by this vector carrying the intact rcsB gene, the gene product RcsB blocks bacterial growth. However, if the rcsB gene is inactivated by the insertion of a foreign DNA fragment, this recombinant plasmid no longer inhibits the growth of E. coli HB101. Although E. coli HB101 is not stably transformed by pERISH2, E. coli K-12 strains such as JM109 and C600 can harbor this vector. Therefore, pERISH2 can be amplified in JM109 and be prepared from this strain in a large quantity using conventional methods. A chromosomal gene library of Klebsiella pneumoniae is constructed easily and efficiently by the utilization of this new cloning vector.

Cloning of cDNA encoding the rice 22 kDa protein of Photosystem II (PSII-S) and analysis of light-induced expression of the gene.

Cloning of rice cDNA encoding the chlorophyll-binding 22 kDa protein of Photosystem II (PSII-S) and the light-induced expression of the gene are reported. One of the light-responsive cDNA clones, isolated by screening with a light-specific subtracted cDNA probe, was shown to encode PSII-S of rice. Genomic Southern analysis suggested that the PSII-S gene, psbS, is a single-copy gene in rice. A brief exposure to red light induced a severalfold increase in the steady state level of PSII-S transcripts in etiolated seedlings. The red light effect was reversed by far-red light, suggesting involvement of phytochrome in the PSII-S gene regulation. Prolonged exposure (3 h) to blue light, however, revealed a much stronger effect than red light on the accumulation of PSII-S transcripts in the etiolated seedlings. In dark-adapted green plants, prolonged exposure to blue light induced re-accumulation of transcripts encoding PSII-S, whereas red light had little effect.

A novel importin alpha from rice, a component involved in the process of nuclear protein transport.

In eukaryotes, nuclear proteins that are transported into nuclei have nuclear localization signals (NLSs), which are recognized by proteins called importin alpha. We isolated a rice cDNA, #61L, and the corresponding gene that encodes a protein, which shows significant homology to the importin alpha. Although the encoded protein had only 23-27% amino acid identity to the importin alphas from various organisms including plants, the fusion protein with glutathione S-transferase showed a specific binding activity to the NLS of SV40 T-antigen. These results suggest that the rice #61L protein is a novel importin alpha in plants.

Radiation therapy for T1,2 glottic carcinoma: impact of overall treatment time on local control.

PURPOSE: Local control probabilities of T1,2 glottic laryngeal cancer were evaluated in relation to dose and fractionation of radiation therapy (RT). MATERIALS AND METHODS: Between 1975 and 1993, 96 T1N0M0 glottic cancers and 32 T2N0M0 glottic cancers were treated with definitive RT. Total RT dose was 60-66 Gy/2 Gy for most of the T1 and T2 tumors, although 10 T2 tumors were treated with hyperfractionation (72-74.4 Gy/1.2 Gy bid). Of the 128 patients, 90 T1 glottic tumors and 30 T2 glottic tumors were followed for > 2 years after treatment. Multivariate analyses using the Cox proportional hazards model and a logistic regression analysis were performed to evaluate the significance of prognostic variables on local control. RESULTS: The 5-year local control probability for T1 tumors was 85%, whereas that for T2 tumors was 71%. Multivariate analyses demonstrated that only overall treatment time (OTT) was a significant variable for local control. Total RT dose, normalized total doses at a fraction size of 2 Gy, and fraction size were not significant. Local control probability of T1 tumors with an OTT of 42-49 days was significantly higher than that of tumors with an OTT of > 49 days (P < 0.02). Only a 1-week interruption of RT, due to holidays, significantly reduced the 5-year local control probability of T1 glottic tumors from 89 to 74% (P < 0.05). CONCLUSIONS: These results indicate that OTT is a significant prognostic factor for local control of T1 glottic tumors.

Effects of five anion channel blockers on thrombin- and ionomycin-activated platelet functions.

The inhibitory effects of anion channel blockers were evaluated on aggregation, intracellular Ca2+ rises, and the production of arachidonic acid metabolites in human platelets. Inhibitors included five anion channel blockers: phloretin, probenecid, pyridoxal phosphate, 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS). The degree of inhibition by each of these agents was dose-dependent on thrombin-activated platelet function. These agents generally had no significant inhibitory effects on ionomycin-activated platelet functions. It is suggested that anion mobilization plays a major role in the receptor-mediated activation of platelet functions, but only a minor role in Ca2+ ionophore-induced platelet activation. It is also suggested that several agents may have properties unrelated to anion channel blockers. Phloretin may be a selective cyclooxygenase inhibitor, and probenecid may inhibit phospholipase A2. DIDS and SITS may interfere with certain aggregation-inducing mechanisms.

Clinical efficacy of brachytherapy combined with external-beam radiotherapy and repeated arterial infusion chemotherapy in patients with unresectable extrahepatic bile duct cancer.

The objective of this study was to evaluate the clinical efficacy of brachytherapy combined with external-beam radiotherapy and repeated arterial infusion chemotherapy in improving stent patency and prognosis in patients with unresectable bile duct cancer as compared with brachytherapy alone. Seventeen patients were treated. Five patients received brachytherapy alone before stent placement. Twelve patients received brachytherapy combined with external-beam radiotherapy (n=5), repeated hepatic arterial infusion chemotherapy using an implanted catheter and port (n=1), or both (n=6). Mean survival was significantly improved in the group that received combined therapy as compared with the group that received brachytherapy alone (16.2 months vs. 4.6 months, p<0.01). Although stent occlusion rates were similar in the two groups (42% vs. 40%), there was a trend towards longer stent patency in the combined therapy group than in the brachytherapy group (22 months vs. 3.6 months, p<0.2). Radiation gastritis necessitating gastrectomy developed in 1 patient who received external-beam radiotherapy at more than 50 Gy. Brachytherapy combined with external-beam radiotherapy and repeated hepatic arterial infusion chemotherapy increases survival compared with brachytherapy alone in patients with unresectable bile duct cancer.