Evaluation of chemical chaperones based on the monitoring of Bip promoter activity and visualization of extracellular vesicles by real-time bioluminescence imaging.

It is known that endoplasmic reticulum (ER) stress in cells and extracellular vesicles (EVs) plays a significant role in cancer cells, therefore the evaluation of compounds that can regulate ER stress and EV secretion would be a suitable system for further screening and development of new drugs. In this study, we evaluated chemical chaperones derived from natural products based on monitoring Bip/GRP78 promoter activity during cancer cell growth, at the level of the single cell, by a bioluminescence microscopy system that had several advantages compared with fluorescence imaging. It was found that several chemical chaperones, such as ferulic acid (FA), silybin, and rutin, affected the activity. We visualized EVs from cancer cells using bioluminescence imaging and showed that several EVs could be observed when using CD63 fused with NanoLuc luciferase, which has a much smaller molecular weight and higher intensity than conventional firefly luciferase. We then examined the effects of the chemical chaperones on EVs from cancer cells by bioluminescence imaging and quantified the expression of CD63 in these EVs. It was found that the chemical chaperones examined in this study affected CD63 levels in EVs. These results showed that imaging at the level of the single cell using bioluminescence is a powerful tool and could be used to evaluate chemical chaperones and EVs from cancer cells. This approach may produce new information in this field when taken together with conventional and classical methods.

Transumbilical arterial embolization of a large dural arteriovenous fistula in a low-birth-weight neonate with congestive heart failure.

PURPOSE: The purpose of this study was to report transumbilical arterial embolization of a large dural arteriovenous fistula (AVF) in a low-birth-weight neonate with congestive heart failure (CHF). CASE PRESENTATION: A female neonate was delivered by cesarean section at 31 weeks of gestation. Her birth weight was 1538 g and Apgar scores were 6 at both 1 and 5 min. Because of dyspnea and retracted respiration immediately after birth, she required mechanical ventilation. Ultrasound revealed right cardiac overload and a large cystic mass at the posterior brain. Magnetic resonance imaging on day 1 showed a large dural AVF (dural sinus malformation with arteriovenous (AV) shunts) at the torcular herophili. Umbilical artery and vein catheterization were performed on the same day for neurointervention. CHF prompted emergency embolization on day 8. The transfemoral arterial route could not be used because of its small size and compromised femoral artery blood flow. Transumbilical arterial embolization shrank the AV shunts markedly, resulting in clinical improvement, thus requiring no further intervention. Follow-up angiography at 4 months confirmed no residual AVF. Her growth and development were normal at the last follow-up at age 4 years. CONCLUSION: This patient apparently was the lowest birth weight neonate with a large AVF successfully treated by embolization, which is usually performed through the transfemoral arterial route. The transumbilical arterial route is an alternative for neonates with birth weight <2000 g and very small femoral arteries.

Combining fluorescence and bioluminescence microscopy.

Bioluminescence microscopy has revealed that gene expression in individual cells can respond differently to the same stimulus. To understand this phenomenon, it is important to sequentially observe the series of events from cellular signal transduction to gene expression regulated by specific transcription factors derived from signaling cascades in individual cells. However, these processes have been separately analyzed with fluorescence and bioluminescence microscopy. Furthermore, in culture medium, the background fluorescence of luciferin-a substrate of luciferase in promoter assays of gene expression in cultured cells-confounds the simultaneous observation of fluorescence and bioluminescence. Therefore, we optimized conditions for optical filter sets based on spectral properties and the luciferin concentration based on cell permeability for fluorescence observation combined with bioluminescence microscopy. An excitation and emission filter set (492-506 nm and 524-578 nm) was suitable for green fluorescent protein and yellow fluorescent protein imaging of cells, and >100 µM luciferin was acceptable in culture medium based on kinetic constants and the estimated intracellular concentration. Using these parameters, we present an example of sequential fluorescence and bioluminescence microscopic observation of signal transduction (translocation of protein kinase C alpha from the cytoplasm to the plasma membrane) coupled with activation of gene expression by nuclear factor of kappa light polypeptide B in individual cells and show that the gene expression response is not completely concordant with upstream signaling following stimulation with phorbol-12-myristate-13-acetate. Our technique is a powerful imaging tool for analysis of heterogeneous gene expression together with upstream signaling in live single cells.

Transfection efficiency of normal and cancer cell lines and monitoring of promoter activity by single-cell bioluminescence imaging.

The bioluminescence system (luciferase reporter assay system) is widely used to study gene expression, signal transduction and other cellular activities. Although transfection of reporter plasmid DNA to mammalian cell lines is an indispensable experimental step, the transfection efficiency of DNA varies among cell lines, and several cell lines are not suitable for this type of assay because of the low transfection efficiency. In this study, we confirm the transfection efficiency of reporter DNA to several cancer and normal cell lines after transient transfection by single-cell imaging. Luminescence images could be obtained from living single cells after transient transfection, and the calculated transfection efficiency of this method was similar to that of the conventional reporter assay using a luminometer. We attempted to measure the activity of the Bip promoter under endoplasmic reticulum stress conditions using both high and low transfection efficiency cells for plasmid DNA at the single-cell level, and observed activation of this promoter even in cells with the lowest transfection efficiency. These results show that bioluminescence imaging of single cells is a powerful tool for the analysis of gene expression based on a reporter assay using limited samples such as clinical specimens or cells from primary culture, and could provide additional information compared with the conventional assay.

Improved method of phosphopeptides enrichment using biphasic phosphate-binding tag/C18 tip for versatile analysis of phosphorylation dynamics.

A number of factors including low stoichiometry of phosphorylation, ion suppression, and reduced peptide backbone fragmentation interfere with precise identification of proteins in phosphoproteomic analysis by MS. Therefore, enrichment of phosphopeptides is an important process for subsequent mass spectrometric analysis. Here, we have developed a simple and efficient method for phosphopeptides enrichment, which employs a biphasic phosphate-binding tag (Phos-tag)/C18 tip consisting of overlaid Phos-tag on the C18 resin in a pipet tip. The improvement in selectivity for phosphopeptides was achieved by using a 40% ACN solution under the phosphopeptides binding conditions. We also assessed the adequacy of Phos-tag/C18 tip for quantitative phosphoproteomic analysis using the iTRAQ technology. After protein digestion and subsequent iTRAQ labeling, interfering substances including excess iTRAQ reagent were directly removed by Phos-tag/C18 tip in a single step. Applying this method, phosphoproteomic analysis of HeLa cells stimulated with tumor necrosis factor -alpha was rapidly and successfully achieved.

Dexmedetomidine exerts dose-dependent age-independent antinociception but age-dependent hypnosis in Fischer rats.

Dexmedetomidine (Dex), an alpha(2)-adrenoceptor agonist, is an effective analgesic and sedative drug in adults; however, little information is available about its efficacy in pediatric populations. Some anesthetics exhibit an age-dependent analgesic effect, e.g., nitrous oxide, being relatively ineffective in newborn rats. We investigated the analgesic and hypnotic efficacy of Dex using 6 cohorts of Fischer rats aged 7, 15, 19, 23, and 29 days and adults exposed to either Dex (10 or 50 microg/kg) or saline subcutaneously. Formalin plantar testing was used to mimic inflammatory pain, and its effect was assessed using immunohistochemical (c-Fos staining) and behavioral methods. The hypnotic action of Dex was assessed by loss of righting reflex. Formalin administration produced a typical nociceptive response in each age group; these nociceptive responses were significantly attenuated by Dex 50 microg/kg at all ages (P < 0.05), whereas Dex 10 microg/kg had little effect. Neonatal rats showed the greatest hypnotic sensitivity to Dex (P < 0.05).

RNA degradation in human breast tissue after surgical removal: a time-course study.

There is much interest in the study of human malignancy using gene expression profiling techniques. Expression profiles obtained from microarrays utilize RNA extracted from the tissue in question. Currently, cell cultures or fresh tissue processed "quickly" are used in these studies. To our knowledge, there are no published reports of a time-course of RNA degradation in surgically removed breast tissue. Such a time-course study is critically needed. We obtained normal breast tissue from breast reduction surgery. Portions of breast tissue kept at room temperature were sampled and placed into RNAlater to preserve RNA at different time-points from 10 min to 3 h after the surgical removal. We evaluated total RNA integrity from each specimen using agarose gel electrophoresis and real-time quantitative RT-PCR analysis of four genes. Electrophoresis showed good-quality, intact RNA at all time points up to 3 h. Quantitative RT-PCR showed no difference in amplified products among all samples. Our study showed that there was no loss of RNA integrity in normal breast tissue for up to 3 h after surgical removal.

Ubiquinol cytochrome c reductase (UQCRFS1) gene amplification in primary breast cancer core biopsy samples.

OBJECTIVES: The ubiquinol cytochrome c reductase UQCRFS1 is a key subunit of the cytochrome bc1 complex (complex III) of the mitochondrial respiratory chain. The purpose of this study is to evaluate the significance of the ubiquinol cytochrome c reductase UQCRFS1 gene amplification in primary breast cancers. METHODS: Samples were obtained from image-guided core needle biopsies (CNB) in 40 patients with nontreated breast cancers. To examine UQCRFS1 gene amplification, we employed fluorescent in situ hybridization using BACs RP11-46I12 that harbors UQCRFS1 gene, RP11-110J19 that overlaps to RP11-46I12, and CA125 as a control gene. The amplification data were evaluated blindly of histopathological factors. RESULTS: Amplification of UQCRFS1 gene was found in 5 of 39 specimens (12.8%). The specimens with amplified UQCRFS1 gene were associated with high grade of cancer cells (P = 0.005). CONCLUSIONS: These results suggest that the UQCRFS1 gene appears to be involved in development of more aggressive phenotype of breast cancer.

Brain stem opioidergic and GABAergic neurons mediate the antinociceptive effect of nitrous oxide in Fischer rats.

BACKGROUND: Recent studies have revealed that N2O exerts its antinociceptive effect by inducing opioid peptide release in the brain stem, thereby activating the descending noradrenergic inhibitory neurons, which modulate pain processing in the spinal cord. However, the precise neuronal pathways that mediate these events remain to be determined. METHODS: Using immunohistochemical and behavioral techniques in adult male Fischer rats, the authors studied the involvement of brain stem opioidergic and gamma-aminobutyric acid-mediated (GABAergic) neurons in the N2O-induced antinociceptive effect using discrete microinjections of an opioid receptor antagonist or GABAergic activator into the periaqueductal gray area and pontine noradrenergic nuclei. They used c-Fos expression as an immunohistochemical mark of neuronal activation induced by N2O and the plantar test as the behavioral paradigm for nociception. RESULTS: Microinjection of either naloxone (an opioid receptor antagonist) or muscimol (a gamma-aminobutyric acid receptor type A agonist) into the ventrolateral periaqueductal gray area inhibited N2O-induced c-Fos expression in the spinal cord and pontine noradrenergic nuclei, particularly in the A7. Microinjection of either naloxone or muscimol into the A7 nuclei also inhibited N2O-induced c-Fos expression in the spinal cord and the N2O-induced antinociceptive effect by the plantar test. CONCLUSIONS: These results support the hypothesis that both opioidergic and GABAergic neurons mediate the antinociceptive effect of N2O at the periaqueductal gray area and A7 in the brain stem. The authors postulate that N2O-induced opioid peptide release leads to inhibition of GABAergic neurons via opioid receptors. The descending noradrenergic inhibitory pathways, which are tonically inhibited by these gamma-aminobutyric acid neurons, are thereby activated (disinhibited) and modulate pain processing in the spinal cord.

Diet and lung cancer risk from a 14-year population-based prospective study in Japan: with special reference to fish consumption.

N-3 polyunsaturated fatty acids in fish oil exhibit a variety of health benefits, and there is evidence that they can inhibit the development of human lung mucoepidermoid and other carcinomas. To examine the hypothesis that fish consumption reduces the risk of lung cancer, we conducted a population-based prospective study, following 5,885 residents for 14 yr. Person-years were used to calculate the relative risk (RR) by the Cox proportional hazards model, with adjustment for potential confounding factors. A total of 51 incident lung cancer cases were observed, and we found linearly decreasing RRs for lung cancer with increased frequency of consumption of fish and shellfish (RRs = 1.00, 0.99, and 0.32, P for trend = 0.003) but not with intake of dried/salted fish. Decreased RRs were apparent with both broiling and boiling cooking methods, but reduction with raw and deep-fried fish consumption was not statistically significant. We conclude that frequent fresh fish consumption, irrespective of the cooking method, may reduce the risk of lung cancer.

Evidence for the involvement of spinal cord alpha1 adrenoceptors in nitrous oxide-induced antinociceptive effects in Fischer rats.

BACKGROUND: In a previous study, the authors found that nitrous oxide (N2O) exposure induces c-Fos (an immunohistochemical marker of neuronal activation) in spinal cord gamma-aminobutyric acid-mediated (GABAergic) neurons in Fischer rats. In this study, the authors sought evidence for the involvement of alpha1 adrenoceptors in the antinociceptive effect of N2O and in activation of GABAergic neurons in the spinal cord. METHODS: Adult male Fischer rats were injected intraperitoneally with alpha1 adrenoceptor antagonist, alpha2 adrenoceptor antagonist, opioid receptor antagonist, or serotonin receptor antagonist and, 15 min later, were exposed to either air (control) or 75% N2O. In some animals, nociception was investigated with the plantar test after 30 min of exposure, while in other animals, gas exposure was continued for 90 min and the spinal cord was examined for c-Fos immunostaining. In a separate experiment, animals were exposed to the above gases alone, after which the spinal cords were examined immunohistochemically for c-Fos and alpha1 adrenoceptor by double-staining methods. RESULTS: The antinociceptive effect of N2O was attenuated by prazosin (an alpha1 adrenoceptor antagonist), yohimbine (an alpha2 adrenoceptor antagonist), and naloxone (an opioid receptor antagonist) but not by methysergide and tropisetron (serotonin receptor antagonists). N2O exposure induced c-Fos expression in the spinal cord, which was blocked by prazosin and naloxone but not by other drugs. N2O-induced c-Fos expression was colocalized with alpha1 adrenoceptor immunoreactivity in laminae III-IV. CONCLUSIONS: These findings support the hypothesis that N2O activates GABAergic interneurons through alpha1 adrenoceptors to produce its antinociceptive effect.

Nitrous oxide exerts age-dependent antinociceptive effects in Fischer rats.

Nitrous oxide (N(2)O) is an inhalational anesthetic/analgesic gas that has been used for clinical practice for more than a century. While its anesthetic mechanisms remain largely unknown, the underlying analgesic mechanisms are now being unraveled. It has been proposed that N(2)O induces opioid peptide release in the midbrain, leading to the activation of descending noradrenergic inhibitory neurons, which modulates pain processing within the spinal cord. Because descending noradrenergic inhibitory neurons are not functional at birth we posit that N(2)O only becomes an effective analgesic/antinociceptive agent in young patients when the descending noradrenergic inhibitory neurons become fully functional. In the present study, we have examined the age-dependence of N(2)O-induced antinociceptive effects on the formalin test. Fischer rats of various ages (7-, 15-, 19-, 23-, and 29-day-old, and adult) were injected 5% formalin into the hind paw during exposure to 75% N(2)O. Both their behavioral responses and changes in Fos-like immunoreactivity in the spinal cord were assessed as markers of N(2)O's antinociceptive effect. Adult-like antinociceptive responses to N(2)O, both behaviorally and immunohistochemically, were only present in rats older than 3 weeks (23- and 29-day-old). These findings support our hypothesis that N(2)O lacks antinociceptive effects in the very young animals.

Activation and suppression of renin-angiotensin system in human dendritic cells.

We previously identified the gene expression of renin-angiotensin system in human monocyte-derived dendritic cells (DCs). This study was conducted to examine the mechanisms by which angiotensin II and captopril, the inhibitor of the angiotensin-converting enzyme (ACE), affect human DCs. In DCs, lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-(IL)-1alpha, IL-10, IL-12, and IL-18 was significantly inhibited by captopril. In contrast, angiotensin II treatment resulted in a significant increase in TNF-alpha and IL-6 protein biosynthesis by DCs. In addition, we have studied the global expression of 2400 genes in DCs from two donors. Here, we demonstrated the specific down-regulation of the ACE gene expression in captopril-treated DCs. Our finding indicates the possible activation of NF-kappaB through the up-regulation of expressions of MEFV gene (encoding PYRIN protein) and heterogeneous nuclear ribonucleoprotein R in DCs. This is the first study on the modulation of cytokine and gene expression by angiotensin II and captopril in DCs.