Induction of apoptosis and necroptosis by 24(S)-hydroxycholesterol is dependent on activity of acyl-CoA:cholesterol acyltransferase 1.

24(S)-hydroxycholesterol (24S-OHC), which is enzymatically produced in the brain, has an important role in maintaining brain cholesterol homeostasis. We have previously reported that 24S-OHC induces necroptosis in human neuroblastoma SH-SY5Y cells. In the present study, we investigated the mechanisms by which 24S-OHC-induced cell death occurs. We found that lipid droplets formed at the early stages in the treatment of SH-SY5Y cells with 24S-OHC. These lipid droplets could be almost completely eliminated by treatment with a specific inhibitor or by siRNA knockdown of acyl-CoA:cholesterol acyltransferase 1 (ACAT1). In association with disappearance of lipid droplets, cell viability was recovered by treatment with the inhibitor or siRNA for ACAT1. Using gas chromatography-mass spectrometry, we confirmed that 24S-OHC-treated cells exhibited accumulation of 24S-OHC esters but not of cholesteryl esters and confirmed that accumulation of 24S-OHC esters was reduced when ACAT1 was inhibited. 24S-OHC induced apoptosis in T-lymphoma Jurkat cells, which endogenously expressed caspase-8, but did not induce apoptosis in SH-SY5Y cells, which expressed no caspase-8. In Jurkat cells treated with the pan-caspase inhibitor ZVAD and in caspase-8-deficient Jurkat cells, 24S-OHC was found to induce caspase-independent cell death, and this was partially but significantly inhibited by Necrostatin-1. Similarly, knockdown of receptor-interacting protein kinase 3, which is one of the essential kinases for necroptosis, significantly suppressed 24S-OHC-induced cell death in Jurkat cells treated with ZVAD. These results suggest that 24S-OHC can induce apoptosis or necroptosis, which of the two is induced being determined by caspase activity. Regardless of the presence or absence of ZVAD, 24S-OHC treatment induced the formation of lipid droplets and cell death in Jurkat cells, and this was suppressed by treatment with ACAT1 inhibitor. Collectively, these results suggest that it is ACAT1-catalyzed 24S-OHC esterification and the resulting lipid droplet formation that is the initial key event which is responsible for 24S-OHC-induced cell death.

Visualization of the leakage of pancreatic juice using a chymotrypsin-activated fluorescent probe.

BACKGROUND: Pancreatic fistula (PF) remains the most serious complication after digestive surgery. It is difficult to prevent because of the inability to visualize the leakage of pancreatic juice during surgery or to evaluate the protease activity of leaked fluid, which is responsible for PF formation. METHODS: The fluorescence intensities of a chymotrypsin probe (glutaryl-phenylalanine [corrected] hydroxymethyl rhodamine green with added trypsin) in pancreatic juice and in intestinal or abdominal fluids drained after pancreatic resection were evaluated. The chymotrypsin probe was sprayed on to filter papers that had been placed on the resected pancreatic stump in patients undergoing pancreaticoduodenectomy or central pancreatectomy. The ability of this technique to visualize the leakage of pancreatic juice and predict postoperative PF formation was assessed. RESULTS: The fluorescence intensity of the chymotrypsin probe in 76 fluid samples correlated positively with amylase levels (r(s) = 0.678, P < 0.001). The fluorescence patterns of the pancreatic stump were classified grossly into the three types: duct (fluorescence signal visualized only on the stump of the main pancreatic duct, 16 patients), diffuse (ductal stump and surrounding pancreatic parenchyma, 7) and negative (no fluorescence signal, 7). Symptomatic PFs developed in 13 of 23 patients with duct- or diffuse-type fluorescence, but in none of the seven patients with negative-type fluorescence (P = 0.008). CONCLUSION: The chymotrypsin probe enabled determination of the protease activity in drained pancreatic fluid samples and allowed real-time visualization of pancreatic juice leakage during surgery.

Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin.

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

Association of human herpesvirus 6 reactivation with the flaring and severity of drug-induced hypersensitivity syndrome.

BACKGROUND: Drug-induced hypersensitivity syndrome (DIHS) is an adverse reaction with clinical signs of fever, rash and internal organ involvement. In the vast majority of patients in Japan, the causative drugs for DIHS are limited to the following eight: carbamazepine, phenytoin, phenobarbital, zonisamide, mexiletine, dapsone, salazosulfapyridine and allopurinol. The association of human herpesvirus (HHV)-6 reactivation with DIHS has been reported by various groups. OBJECTIVES: To confirm the relationship between the flaring and severity of DIHS and HHV-6 reactivation. METHODS: We evaluated 100 patients with drug rash and systemic symptom(s) caused by the drugs associated with DIHS. HHV-6 reactivation was examined by serological antibody assay and quantitative real-time polymerase chain reaction assay of serial serum samples. RESULTS: Anti-HHV-6 IgG titres increased in 62 of 100 patients, 14-28 days after the onset of symptoms. These patients suffered from severe organ involvement and a prolonged course compared with 38 patients showing no reactivation of HHV-6. Significant amounts of HHV-6 DNA were detected in serum samples from 18 of the 62 patients. Flaring of symptoms such as fever and hepatitis was closely related to HHV-6 reactivation in these 18 patients. It should be emphasized that all five patients with fatal outcome and 10 patients with renal failure were in the HHV-6 reactivation group. CONCLUSIONS: A combination of immunological reaction to a drug and HHV-6 reactivation results in the severe course of DIHS. The demonstration of HHV-6 reactivation is a useful marker of diagnosis as well as prognosis in DIHS.

Investigation of the origin of ephedrine and methamphetamine by stable isotope ratio mass spectrometry: a Japanese experience.

Illicit drug abuse is a serious global problem that can only be solved through international cooperation. In Asian countries, the abuse of methamphetamine is one of the most pressing problems. To assist in the control of methamphetamine, the authors investigated in detail the character of ephedrine, which is a key precursor for the illicit manufacture of methamphetamine. Commercial ephedrine is produced by one of three methods: (a) extraction from Ephedra plants, (b) full chemical synthesis or (c) via a semi-synthetic process involving the fermentation of sugar, followed by amination. Although chemically there is no difference between ephedrine samples from different origins (natural, synthetic or semi-synthetic), scientific and analytical tools such as drug-characterization and impurity-profiling programmes may provide valuable information for law enforcement and regulatory activities as part of precursor control strategies. During the research under discussion in the present article, in addition to classical impurity profiling of manufacturing by-products, the use of stable isotope ratio mass spectrometry was investigated for determining the origin of the ephedrine that had been used as a precursor in seized methamphetamine samples. The results of carbon and nitrogen stable isotope ratio (delta13C and delta15N) analysis of samples of crystalline methamphetamine seized in Japan suggested that the drug had been synthesized from either natural or semi-synthetic ephedrine and not from synthetic ephedrine. Stable isotope ratio analysis is expected to be a useful tool for tracing the origins of seized methamphetamine. It has attracted much interest from precursor control authorities in Japan and the East Asian region and may prove useful in the international control of precursors.

Amyloid beta induces neuronal cell death through ROS-mediated ASK1 activation.

Amyloid beta (Abeta) is a main component of senile plaques in Alzheimer's disease and induces neuronal cell death. Reactive oxygen species (ROS), nitric oxide and endoplasmic reticulum (ER) stress have been implicated in Abeta-induced neurotoxicity. We have reported that apoptosis signal-regulating kinase 1 (ASK1) is required for ROS- and ER stress-induced JNK activation and apoptosis. Here we show the involvement of ASK1 in Abeta-induced neuronal cell death. Abeta activated ASK1 mainly through production of ROS but not through ER stress in cultured neuronal cells. Importantly, ASK1-/- neurons were defective in Abeta-induced JNK activation and cell death. These results indicate that ROS-mediated ASK1 activation is a key mechanism for Abeta-induced neurotoxicity, which plays a central role in Alzheimer's disease.

M6a acts as a nerve growth factor-gated Ca(2+) channel in neuronal differentiation.

To elucidate the function of M6a, which is a neuron-specific membrane glycoprotein of the brain and possesses putative phosphorylation sites for protein kinase C (PKC), we established rat M6a cDNA expression vector-transfected PC12 cells. These transfectants exhibited high susceptibilities to nerve growth factor (NGF) for neuronal differentiation. Interestingly, we found that Ca(2+) influx in these transfectants was significantly augmented by the treatment of NGF, but not epidermal growth factor (EGF), which stimulates PC12 cell growth. NGF-dependent augmentation of Ca(2+) influx was detected within 3h and severely inhibited by EGTA- and PKC-specific inhibitors. Anti-M6 antibody suppressed both NGF-triggered Ca(2+) influx and neuronal differentiation. These results support the idea that M6a implicates in neuronal differentiation as a novel Ca(2+) channel gated selectively by phosphorylation with PKC in the downstream of NGF signaling pathway.

FR226807: a potent and selective phosphodiesterase type 5 inhibitor.

We describe the pharmacological characteristics of a novel phosphodiesterase type 5 inhibitor FR226807, N-(3,4-dimethoxybenzyl)-2-[[(1R)-2-hydroxy-1-methylethyl]amino]-5-nitrobenzamide. FR226807 inhibited phosphodiesterase type 5 isolated from human platelets with an IC(50) value of 1.1 nM. FR226807 also inhibited phosphodiesterase type 6 with an IC(50) of 20 nM; however, the IC(50) value for phosphodiesterase type 6 was 18-fold higher than that for phosphodiesterase type 5. The IC(50) values of FR226807 for other phosphodiesterases (phosphodiesterase type 1, phosphodiesterase type 2, phosphodiesterase type 3, and phosphodiesterase type 4) were 1000-fold higher than that for phosphodiesterase type 5. FR226807 increased the cyclic guanosine monophosphate (cGMP) content in corpus cavernosum isolated from rabbit, an effect associated with relaxation of the muscle. FR226807 enhanced the relaxation response induced by electrical field stimulation of corpus cavernosum isolated from the rabbit. In an anesthetized dog model for the evaluation of erectile function, intravenous administration of FR226807 prolonged the time to return to 75% of maximal intracavernosal pressure after cessation of electrical stimulation of the pelvic nerve. In summary, FR226807 is a potent and highly selective phosphodiesterase type 5 inhibitor with an augmentative effect on penile erection and will be useful for the treatment of erectile dysfunction.

Rational design of fluorescein-based fluorescence probes. Mechanism-based design of a maximum fluorescence probe for singlet oxygen.

Fluorescein is one of the best available fluorophores for biological applications, but the factors that control its fluorescence properties are not fully established. Thus, we initiated a study aimed at providing a strategy for rational design of functional fluorescence probes bearing fluorescein structure. We have synthesized various kinds of fluorescein derivatives and examined the relationship between their fluorescence properties and the highest occupied molecular orbital (HOMO) levels of their benzoic acid moieties obtained by semiempirical PM3 calculations. It was concluded that the fluorescence properties of fluorescein derivatives are controlled by a photoinduced electron transfer (PET) process from the benzoic acid moiety to the xanthene ring and that the threshold of fluorescence OFF/ON switching lies around -8.9 eV for the HOMO level of the benzoic acid moiety. This information provides the basis for a practical strategy for rational design of functional fluorescence probes to detect certain biomolecules. We used this approach to design and synthesize 9-[2-(3-carboxy-9,10-dimethyl)anthryl]-6-hydroxy-3H-xanthen-3-one (DMAX) as a singlet oxygen probe and confirmed that it is the most sensitive probe currently known for (1)O(2). This novel fluorescence probe has a 9,10-dimethylanthracene moiety as an extremely fast chemical trap of (1)O(2). As was expected from PM3 calculations, DMAX scarcely fluoresces, while DMAX endoperoxide (DMAX-EP) is strongly fluorescent. Further, DMAX reacts with (1)O(2) more rapidly, and its sensitivity is 53-fold higher than that of 9-[2-(3-carboxy-9,10-diphenyl)anthryl]-6-hydroxy-3H-xanthen-3-ones (DPAXs), which are a series of fluorescence probes for singlet oxygen that we recently developed. DMAX should be useful as a fluorescence probe for detecting (1)O(2) in a variety of biological systems.

Germline mutations of the PTCH gene in Japanese patients with nevoid basal cell carcinoma syndrome.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental and skeletal anomalies, palmo-plantar pits, odontogenic keratocysts, ectopic calcification, and occurrence of various types of tumors including basal cell carcinoma. Recent evidence has indicated that the human homologue of a Drosophila segment polarity gene, PTCH, is a NBCCS susceptibility gene. In the study presented here, we detected two novel mutations of the PTCH gene, I805X/2395delC and Y93X/C297A, in two unrelated Japanese patients. Early protection of the skin from the sunlight is important to the prevention of BCC development in NBCCS patients. Genetic analysis of the PTCH gene is essential for the early, definitive diagnosis of NBCCS, especially before the expression of clinical manifestations is complete.

Bioimaging of nitric oxide with fluorescent indicators based on the rhodamine chromophore.

Diaminofluoresceins are widely used for detection and imaging of nitric oxide (NO), but for biological applications, they have the disadvantages that the fluorescence of the fluorescein chromophore is pH-sensitive and overlaps the autofluorescence of cells. We have developed a membrane-permeable fluorescent indicator for NO based on the rhodamine chromophore, DAR-4M AM, which can be excited with 550-nm light. The fluorescence quantum yield of the product after reaction with NO is 840 times higher than that of DAR-4M. The detection limit of NO was 7 nM, and the fluorescence showed no pH dependency above pH 4. DAR-4M AM was successfully applied to practical bioimaging of NO produced in bovine aortic endothelial cells.

Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides.

The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the alpha carbon atom. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay.

Intramolecular fluorescence resonance energy transfer system with coumarin donor included in beta-cyclodextrin.

In aqueous solutions, the fluorescence of the intramolecular fluorescence resonance energy-transfer (FRET) system 1 was strongly quenched, because of close contact between the donor and acceptor moieties. FRET occurred, and the acceptor fluorescence was increased, by adding beta-cyclodextrin (beta-CD) to aqueous solutions of 1. Spectral analysis supported the idea that the FRET enhancement was due to the formation of an inclusion complex of the coumarin moiety in beta-CD, resulting in separation of the fluorophores. On the basis of this result, we propose that covalent binding of coumarin to beta-CD will provide a FRET cassette molecule. So, compound 2 bearing beta-CD covalently was designed and synthesized. Fluorescence intensity of 2 was enhanced markedly compared to the intensity of 3. Applying this FRET system, various FRET probes that will be useful for ratio imaging and also the high-throughput screening will be provided.

Prediction of flammability of gases by using F-number analysis.

A novel method of predicting flammability limits has been proposed. This method utilizes a new flammability index called F-number. For this purpose, an empirical expression of F-number has been derived to account for the flammability characteristics of various organic substances. The analysis has been done by fitting to the observed values of F-number for a wide variety of organic gases and vapors. As a result, it has been found that F-number is an excellent tool to analyze the flammability characteristics of various substances. It has also been shown that the values of upper and lower flammability limits can be derived from F-number together with the stoichiometric concentration corrected for the effect of selective diffusion.

The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells.

The effect of cultured normal human dermal papilla cells (DPCs) and conditioned medium prepared with cultured DPCs on chemotactic migration of human hair outer root sheath cells (ORSCs) was examined quantitatively. ORSCs showed significantly increased migration toward both cultured DPCs and the conditioned medium suggesting that DPCs produce and secrete a paracrine factor(s), which attracts hair follicle epithelial cells. Some soluble factors, which are reportedly produced by DPCs, such as insulin-like growth factor-I (IGF-I), hepatocyte growth factor (HGF), vascular endothelial cell growth factor (VEGF), and transforming growth factor-beta1 (TGF-beta1), were also examined. ORSCs showed dramatically increased migration toward IGF-I and HGF at concentrations of 1-10 ng/ml. On the other hand, neither VEGF nor TGF-beta1 showed any effect on the chemotaxis of ORSCs. It is interesting that all factors involving mitogenic activity did not always have chemotactic activity for ORSCs. This is the first report to establish that IGF-I and HGF have not only a growth stimulatory but also a chemotactic effect on ORSCs. In addition, the method presented here may help to simplify chemotaxis assays of any type of epithelial keratinocytes with poor mobility.

Molecular cloning and functional characterization of cDNAs encoding cysteine synthase and serine acetyltransferase that may be responsible for high cellular cysteine content in Allium tuberosum.

The plants belonging to the genus Allium are known to accumulate sulfur-containing secondary compounds that are derived from cysteine. Here, we report on molecular cloning and functional characterization of two cDNAs that encode serine acetyltransferase and cysteine synthase from A. tuberosum (Chinese chive). The cDNA for serine acetyltransferase encodes an open reading frame of 289 amino acids, of which expression could complement the lacking of cysE gene for endogenous serine acetyltransferase in Escherichia coli. The cDNA for cysteine synthase encodes an open reading frame of 325 amino acids, of which expression in the E. coli lacking endogenous cysteine synthase genes could functionally rescue the growth without addition of cysteine. Both deduced proteins seem to be localized in cytosol, judging from their primary structures. Northern blot analysis indicated that both transcripts accumulated in almost equal levels in leaves and root of green and etiolated seedlings of A. tuberosum. The activity of recombinant serine acetyltransferase produced from the cDNA was inhibited by L-cysteine, which is the end-product of the pathway; however, the sensitivity to cysteine (48.7 microM of the concentration for 50% inhibition, IC(50)) was fairly low compared with that of previously reported serine acetyltransferases ( approximately 5 microM IC(50)) from various plants. In A. tuberosum, the cellular content of cysteine was several-fold higher than those in Arabidopsis thaliana and tobacco. This higher concentration of cysteine in A. tuberosum is likely due to the lower sensitivity of feedback inhibition of serine acetyltransferase to cysteine.

Superoxide dismutase activity of iron(II)TPEN complex and its derivatives.

Superoxide is involved in the pathogenesis of various diseases, such as inflammation, ischemia-reperfusion injury and carcinogenesis. Superoxide dismutases (SODs) catalyze the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, and can protect living cells against the toxicity of free radicals derived from oxygen. Thus, SODs and their functional mimics have potential value as pharmaceuticals. We have previously reported that Fe(II)tetrakis-N,N,N',N'-(2-pyridylmethyl)ethylenediamine (Fe(II)TPEN) has an excellent SOD activity (IC50 = 0.5 microM) among many iron complexes examined (J. Biol. Chem., 264, 9243-9249 (1989)). Fe(II)TPEN can act like native SOD in living cells, and protect Escherichia coli cells from free radical toxicity caused by paraquat. In order to develop more effective SOD functional mimics, we synthesized Fe(II)TPEN derivatives with electron-donating or electron-withdrawing groups at the 4-position of all pyridines of TPEN, and measured the SOD activities and the redox potentials of these complexes. Fe(II) tetrakis-N,N,N',N'-(4-methoxy-2-pyridylmethyl)ethylenediamine (Fe(II)(4MeO)4TPEN) had the highest SOD activity (IC50 = 0.1 microM) among these iron-based SOD mimics. In addition, a good correlation was found between the redox potential and the SOD activity of 15 Fe(II) complexes, including iron-based SOD mimics reported in the previous paper (J. Organometal. Chem., in press). Iron-based SOD mimics may be clinically applicable, because these complexes are generally tissue-permeable and show low toxicity. Therefore our findings should be significant for the development of clinically useful SOD mimics.

Development of a time-resolved fluorometric detection system using diffusion-enhanced energy transfer

A novel detection system using both emission energy transfer and time-resolved fluorometry (TRF) was developed, with a europium chelate as the energy donor and a novel fluorophore SNR1, excitable with long-wavelength light corresponding to europium emission, as the energy acceptor. When the donor and acceptor molecules were mixed in solution, energy transfer was observed without direct attachment of the donor and the acceptor, via a diffusion-enhanced energy-transfer mechanism. Thus, the acceptor emission can be detected as a long-lifetime fluorescence in TRF. When the fluorescence properties of the acceptor molecule are changed by interaction with an enzyme or other bioactive molecule, the change can be detected as a long-lived sensitized emission. If we develop or select suitable acceptor molecules, this simple and convenient system should be applicable to a wide variety of bioactive molecules. Since it is based on TRF, it can be used for high-resolution assay.