Estimating age-related changes in in vivo cerebral magnetic resonance angiography using convolutional neural network.

Although age-related changes of cerebral arteries were observed in in vivo magnetic resonance angiography (MRA), standard tools or methods measuring those changes were limited. In this study, we developed and evaluated a model to measure age-related changes in the cerebral arteries from 3D MRA using a 3D deep convolutional neural network. From participants without any medical abnormality, training (n = 800) and validation sets (n = 88) of 3D MRA were built. After preprocessing and data augmentation, a 3D convolutional neural network was trained to estimate each subject's chronological age from in vivo MRA data. There was good correlation between chronological age and predicted age (r = 0.83) in an independent test set (n = 354). The predicted age difference (PAD) of the test set was 2.41 ± 6.22. Interaction term between age and sex was significant for PAD (p = 0.008). After correcting for age and interaction term, men showed higher PAD (p < 0.001). Hypertension was associated with higher PAD with marginal significance (p = 0.073). We suggested that PAD might be a potential measurement of cerebral vascular aging.

Regulation of RANKL-Induced Osteoclastogenesis by 635-nm Light-Emitting Diode Irradiation Via HSP27 in Bone Marrow-Derived Macrophages.

OBJECTIVE: This study was designed to investigate the effect of 635-nm irradiation from a light-emitting diode (LED) on osteoclastogenesis in receptor activator of nuclear factor kappa-B (NF-κB) ligand (RANKL)-stimulated mouse bone marrow-derived macrophages (BMMs). We determined whether 635-nm irradiation modulated the RANKL-induced osteoclastic signaling pathway in heat shock protein-27 (HSP27)-silenced cells and analyzed the functional cross talk between these factors in osteoclastic differentiation and activation. BACKGROUND: HSP27, a member of the small HSP family, regulates oxidative stress. Clinical reports suggest that low-level laser therapy or LED therapy (LEDT) could be an effective alternative treatment for osteolytic bone disease. METHODS: In control or HSP27-siRNA-treated BMMs, the effects of LED irradiation with 635 nm and 5 mW/cm2 on RANKL-induced osteoclastic differentiation and activity were assessed by measuring tartrate-resistant acid phosphatase (TRAP) and resorption pit formation. Quantitative real-time polymerase chain reaction and western blot assays were carried out to assess the mRNA expression of osteoclastogenesis-related genes and phosphorylation of c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), AKT, and p38, respectively. Intracellular reactive oxygen species (ROS) generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) detection method. RESULTS: The 635-nm irradiation treatment significantly increased HSP27 expression and decreased intracellular ROS generation, as well as p38 and AKT phosphorylation, leading to reductions in the expression of c-fos, NFATc1, and DC-STAMP and TRAP activation and osteoclastic bone resorption in RANKL-induced BMMs. However, in HSP27-silenced BMMs, no change was observed. CONCLUSIONS: Thus, 635-nm irradiation modulates RANKL-induced osteoclastogenesis via HSP27 in BMMs. Thus, HSP27 may play a role in regulating the osteoclastic response to LEDT.

Zinc(II) ion promotes anti-inflammatory effects of rhSOD3 by increasing cellular association.

Recently, we demonstrated that superoxide dismutase 3 (SOD3) is a strong candidate for biomedicine. Anti-oxidant function of SOD3 was accomplished without cell penetration, and it inhibited the inflammatory responses via non-enzymatic functions. SOD3 has the heparin binding domain associating cell surface. Interestingly, we found that Zn2＋ promotes transduction effects of recombinant human SOD3 (rhSOD3) by increasing uptake via the heparin binding domain (HBD). We demonstrated an uptake of rhSOD3 from media to cell lysate via HBD, resulting in an accumulation of rhSOD3 in the nucleus, which was promoted by the presence of Zn2＋. This resulted in increased inhibitory effects of rhSOD3 on NF-kB and STAT3 signals in the presence of Zn2＋, which shows elevated association of rhSOD3 into the cells. These results suggest that an optimized procedure can help to enhance the inflammatory efficacy of rhSOD3, as a novel biomedicine. [BMB Reports 2017; 50(2): 85-90].

Endothelium- and smooth muscle-dependent vasodilator effects of Citrus aurantium L. var. amara: Focus on Ca(2+) modulation.

Neroli, the essential oil of Citrus aurantium L. var. amara, is a well-characterized alleviative agent used to treat cardiovascular symptoms. However, because it has been found to have multiple effects, its mechanism of action requires further exploration. We sought to clarify the mechanism underlying the actions of neroli in mouse aorta. In aortic rings from mice precontracted with prostaglandin F2 alpha, neroli induced vasodilation. However, relaxation effect of neroli was decreased in endothelium-denuded ring or pre-incubation with the nitric oxide synthase inhibitor NG-Nitro-l-arginine-methyl ester (L-NAME). And also, neroli-induced relaxation was also partially reversed by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ), a soluble guanylyl cyclase (sGC) inhibitor. In addition, neroli inhibited extracellular Ca(2+)-dependent, depolarization-induced contraction, an effect that was concentration dependent. Pretreatment with the non-selective cation channel blocker, Ni(2+), attenuated neroli-induced relaxation, whereas the K(+) channel blocker, tetraethylammonium chloride, had no effect. In the presence of verapamil, added to prevent Ca(2+) influx via smooth muscle voltage-gated Ca(2+) channels, neroli-induced relaxation was reduced by the ryanodine receptor (RyR) inhibitor ruthenium red. Our findings further indicate that the endothelial component of neroli-induced vasodilation is partly mediated by the NO-sGC pathway, whereas the smooth muscle component involves modulation of intracellular Ca(2+) concentration through inhibition of cation channel-mediated extracellular Ca(2+) influx and store-operated Ca(2+) release mediated by the RyR signaling pathway.

Evaluation of general toxicity and genotoxicity of the silkworm extract powder.

The silkworm extract powder contain 1-deoxynojirimycin (DNJ), a potent α-glycosidase inhibitor, has therapeutic potency against diabetes mellitus. Therefore, natural products containing DNJ from mulberry leaves and silkworm are consumed as health functional food. The present study was performed to evaluate the safety of the silkworm extract powder, a health food which containing the DNJ. The repeated toxicity studies and gentic toxicity studies of the silkworm extract powder were performed to obtain the data for new functional food approval in MFDS. The safety was evaluated by a single-dose oral toxicity study and a 90 day repeated-dose oral toxicity study in Sprague-Dawley rats. The silkworm extract powder was also evaluated for its mutagenic potential in a battery of genetic toxicity test: in vitro bacterial reverse mutation assay, in vitro chromosomal aberration test, and in vivo mouse bone marrow micronucleus assay. The results of the genetic toxicology assays were negative in all of the assays. The approximate lethal dose in single oral dose toxicity study was considered to be higher than 5000 mg/kg in rats. In the 90 day study, the dose levels were wet at 0, 500, 1000, 2000 mg/kg/day, and 10 animals/sex/dose were treated with oral gavage. The parameters that were monitored were clinical signs, body weights, food and water consumptions, ophthalmic examination, urinalysis, hematology, serum biochemistry, necropsy findings, organ weights, and histopathological examination. No adverse effects were observed after the 90 day administration of the silkworm extract powder. The No-Observed-Adverse-Effect-Level (NOAEL) of silkworm extract powder in the 90 day study was 2000 mg/kg/day in both sexes, and no target organ was identified.

Effects of male silkworm pupa powder on the erectile dysfunction by chronic ethanol consumption in rats.

Erectile dysfunction (ED) is a highly prevalent disorder that affects millions of men worldwide. ED is now considered an early manifestation of atherosclerosis, and consequently, a precursor of systemic vascular disease. This study was designed to investigate the effects of male silkworm pupa powder (SWP) on the levels of nitric oxide synthase (NOS) expression, nitrite, and glutathione (GSH); lipid peroxidation; libido; and erectile response of the corpus cavernosum of the rat penis. We induced ED in the study animals by oral administration of 20% ethanol over 8 weeks. The SWP-treated male rats were divided into 3 groups that were orally administered 200, 400, and 800 mg/kg. The libido of the SWP-administered male rats was higher than that of the ethanol control group. In addition, the erectile response of the corpus cavernosum was restored in males on SWP administration, to a level similar to that of the normal group without ED. The testosterone concentration did not increase significantly. The lipid peroxidation in the corpus cavernosum of the male rats administered SWP decreased significantly. In contrast, compared to the ethanol group, SWP-administered male rats showed increased GSH levels in the corpus cavernosum. The level of nitrite and NOS expression in the corpus cavernosum of SWP-administered male rats increased significantly. These results indicated that SWP effectively restored ethanol-induced ED in male rats.

Increased yield of high-purity and active tetrameric recombinant human EC-SOD by solid phase refolding.

Superoxide dismutase (SOD) removes damaging reactive oxygen species from the cellular environment by catalyzing the dismutation of two superoxide radicals to hydrogen peroxide and oxygen. Extracellular superoxide dismutase (EC-SOD) is a tetramer and is present in the extracellular space and to a lesser extent in the extracellular fluids. Increasing therapeutic applications for recombinant human extracellular superoxide dismutase (rEC-SOD) has broadened interest in optimizing methods for its purification, with a native conformation of tetramer. We describe a solid phase refolding procedure that combines immobilized metal affinity chromatography (IMAC) and gel filtration chromatography in the purification of rEC-SOD from Escherichia coli. The purified rEC-SOD tetramer from the Ni(2+)-column chromatography is refolded in Tris buffer. This method yields greater than 90% of the tetramer form. Greater than 99% purity is achieved with further purification over a Superose 12PC 3.2/30 column to obtain the tetramer and specific activities as determined via DCFHDA assay. The improved yield of rEC-SOD in a simple chromatographic purification procedure promises to enhance the development and therapeutic application of this biologically potent molecule.

Expression in yeast of secreted lignin peroxidase with improved 2,4-dichlorophenol degradability by DNA shuffling.

Lignin peroxidase (LiP) from Phanerochaete chrysosporium was shown to mineralize a variety of recalcitrant aromatic compounds and oxidize a number of polycyclic aromatic and phenolic compounds. The major problem of the wild type LiP is that it can be inactivated by excess H(2)O(2) and high concentrations of aromatic compounds. We applied a directed evolution technique coupled with a rapid colorimetric screening method to obtain mutant genes with improved H(2)O(2) stability and polychlorinated phenol degradability, and they were successfully expressed as the secretive LiPs in recombinant Saccharomyces cerevisiae. The resulting variants showed approximately 1.6-fold improved 2,4-dichlorophenol (2,4-DCP) degradation activity and stability against H(2)O(2) compared with the parent strain. The kinetic properties of the variants toward 2,4-DCP and H(2)O(2) were also increased compared with the wild type for all three mutants studied. Amino acid sequence analysis indicated that the greatest number of amino acid substitutions was located near the surface or Ca(2+) binding sites of the enzyme.

Purification of a dimethyladenosine compound from silkworm pupae as a vasorelaxation substance.

To identify the active substance in the male silkworm pupae that strengthens men's vitality, the vasorelaxation activity was determined by measuring the vascular endothelial nitric oxide (eNO) produced in calf pulmonary artery endothelial (CPAE) cells treated with extracts from the pupae. Dried silkworm male pupae were extracted with ethanol and suspended in water, then partitioned with hexane, chloroform, ethylacetate, and butanol, sequentially. Among these fractions, the aqueous fraction had maximal NO production (156.87 microM/200 microl well, 10 mg/ml) and minimal cytotoxicity (IC50 362.3 mg/ml). The vasorelaxation substances (VAS) from the aqueous fraction were isolated by a combination of gel filtration and anion-exchange chromatography on DEAE Sephadex A-25 and reverse phase-HPLC. Their chemical structures were determined on the basis of their spectroscopic parameters of EI-MS, MALDI-TOF MS, 1H and 13C NMR, 1H-1H COSY, and GC-MS spectral data. The active substance was subsequently identified as a dimethyladenosine and dimethyladenosine-5'-L-arabinose that has phosphodiesterase (PDE) inhibition activity. This compound was shown to inhibit PDE4 activity in a dose-dependent manner. Also, it inhibited the PDE5 activity of cyclic-GMP-specific PDE5 enzyme. These results imply that dimethyladenosine may be a lead compound for the development and improvement of vasculogenic impotence drugs through phosphodiesterase inhibition and NO production in endothelial cells.

Functionality improvement of fungal lignin peroxidase by DNA shuffling for 2,4-dichlorophenol degradability and H2O2 stability.

One of the major problems of wild-type lignin peroxidase (LiP) is its inactivity at the presence of excess H(2)O(2) and high concentration of aromatic compounds. Little is known about the substrate-binding site of LiP, and functionality improvement of LiP was not actively tried by genetic engineering and directed evolution. In order to improve LiPs functionality, we performed directed evolution with a colorimetric screening method. Finally, three types of LiP mutants were screened. The catalytic efficiency of the variants toward 2,4-dichlorophenol (DCP) degradation activity and the stability against H(2)O(2) was increased over the wild type. The K(m) value of the variants toward H(2)O(2) was increased, but K(m) value toward 2,4-DCP degradation was reduced. Overall, The K(cat)/K(m) values of the mutants toward 2,4-DCP was increased ca. 4-fold, and that toward H(2)O(2) was increased ca. 89-fold. Amino acid sequence analysis indicated that the most of the mutations were located on the enzyme surface. We expect that these results coupled with recombining mutation can be successfully applied to the molecular evolution cycles for screening of LiPs and other oxidative enzymes with improved functionality and stability.

Anti-hypertensive effect of the Dongchunghacho, Isaria sinclairii, in the spontaneously hypertensive rats.

The present study examined the effect of the methanol extract of Isaria sinclairii, a kind of Donchunghacho (Tochukaso), on blood pressure in spontaneously hypertensive rats (SHR). Blood pressure and heart rate were measured after treatment with the methanol extract of I. sinclairii by the indirect tail-cuff method and the direct in vivo model. Starting at 12 weeks of age, male SHR were treated with the extracts for 2 or 4 weeks. We found that, when compared to untreated control SHR, oral treatment with I. sinclairii methanol extract (30 mg/kg/day) remarkably decreased systolic blood pressure from 200 to 112 mmHg and decreased diastolic blood pressure from 114 to 88 mmHg. Furthermore, efficacy of methanol extract of I. sinclairii was superior to captopril (30 mg/kg/mL, positive control), an angiotensin-converting enzyme inhibitor, with a lowering effect that dropped systolic blood pressure from 201 to 130 mmHg and diastolic blood pressure from 102 to 92 mmHg. However, in normal Wistar Kyoto rats, I. sinclairii methanol extract did not significantly change the normal blood pressure, suggesting that this type of Dongchunghacho has a selective effect against hypertension. Therefore, methanol extract of I. sinclairii may be used as an anti-hypertensive food/agent. Furthermore, this extract also has multiple actions such as No production in endothelial cells, inhibiting thrombin-induced blood coagulation by thrombin and mildly decreasing in prostaglandin E2 levels in cultured macrophage cells, all of which might contribute to protection against atherogenesis and thrombus formation. HPLC and MS analysis of methanol extract of I. sinclairii revealed the presence of adenosine.

Distinct kinetic mechanisms of the two classes of Aminoacyl-tRNA synthetases.

Aminoacyl-tRNA synthetases are divided into two classes based on both functional and structural criteria. Distinctions between the classes have heretofore been based on general features, such as the position of aminoacylation on the 3'-terminal tRNA ribose, and the topology and tRNA-binding orientation of the active-site protein fold. Here we show instead that transient burst kinetics provides a distinct mechanistic signature dividing the two classes of tRNA synthetases, and that this distinction has significant downstream effects on protein synthesis. Steady-state and transient kinetic analyses of class I CysRS and ValRS, and class II AlaRS and ProRS, reveal that class I tRNA synthetases are rate-limited by release of aminoacyl-tRNA, while class II synthetases are limited by a step prior to aminoacyl transfer. The tight aminoacyl-tRNA product binding by class I enzymes correlates with the ability of EF-Tu to form a ternary complex with class I but not class II synthetases, and the further capacity of this protein to enhance the rate of aminoacylation by class I synthetases. These results emphasize that the distinct mechanistic signatures of class I versus class II tRNA synthetases ensure rapid turnover of aminoacyl-tRNAs during protein synthesis.

Crystallization and preliminary X-ray crystallographic analysis of the alpha-2,6-sialyltransferase PM0188 from Pasteurella multosida.

Sialyltransferase is an enzyme that transfers the sialic acid moiety from cytidine-5-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to the carbohydrate group of various glycoproteins. These glycoproteins are involved in inflammation, embryogenesis, immune defence and metastasis of cancer cells by cell-cell interactions or cell-matrix interactions. The alpha-2,6-sialyltransferase PM0188 from Pasteurella multocida was purified using affinity-column chromatographic methods and crystallized using the hanging-drop vapour-diffusion method at 293 K. MAD data were collected to 1.9 A resolution from an SeMet-substituted crystal. The crystal belongs to space group P2(1), with unit-cell parameters a = 52.9, b = 61.0, c = 64.6 A, alpha = gamma = 90, beta = 112.3 degrees. Assuming the presence of one molecule in the asymmetric unit, the solvent content is estimated to be about 45%.

Synthesis of complex carbohydrates and glyconjugates: enzymatic synthesis of globotetraose using alpha-1,3-N-acetylgalactosaminyltransferase LgtD from Haemophilus infuenzae strain Rd.

The lipopolysaccharide of capsule-deficient Haemophilus infuenzae strain Rd contains an N-acetylgalactosamine residue attached to the terminal globotriose moiety in the Hex5 glycoform. Genome analysis identified an open reading frame, HI1578, referred to as LgtD, whose amino acid sequence shows a significant level of similarity to those of a number of bacterial glycosyltransferases involved in lipopolysaccharide biosynthesis. To investigate its function, overexpression and biochemical characterization were performed. Most of the protein was obtained in a highly soluble and active form. Standard glycosyltransferase assay, high-performance liquid chromatography (HPLC), and liquid chromatography (LC)/mass spectrometry (MS) show that LgtD is an N-acetylgalactosaminyltransferase with high donor substrate specificity, and globotriose is a highly preferred acceptor substrate for the enzyme.

Genotoxic evaluation of the biocomponents of the cricket, Gryllus bimaculatus, using three mutagenicity tests.

The mutagenic potential of the extracted components of Gryllus bimaculatus, a species of cricket, was evaluated using short-term genotoxicity tests including the Ames, chromosome aberration, and micronuclei tests. In a Salmonella typhimurium assay, G. bimaculatus extract did not produce any mutagenic response in the absence or presence of S9 mix with TA98, TA100, TA1535, and TA1537. Chromosome aberration testing showed that G. bimaculatus had no significant effect on Chinese hamster ovary (CHO) cells. In the mouse micronucleus test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice intraperitoneally administered with G. bimaculatus extract at doses of 15, 150, or 1500 mg/kg. These results indicate that G. bimaculatus extract exerts no mutagenic effect in these in vitro and in vivo systems.

Purification and characterization of a serine protease (CPM-2) with fibrinolytic activity from the dung beetles.

Catharsius protease-2 (CPM-2) was isolated from the body of dung beetles, Catharsius molossus, using a three step purification process (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and affinity chromatography on DEAE Affi-Gel blue). The purified CPM-2, having a molecular weight of 24 kDa, was assessed homogeneously by SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CPM-2 was composed of X Val Gln Asp Phe Val Glu Glu Ile Leu. CPM-2 was inactivated by Cu2+ and Zn2+ and strongly inhibited by typical serine proteinase inhibitors such as TLCK, soybean trypsin inhibitor, aprotinin, benzamidine, and alpha1-antitrypsin. However, EDTA, EGTA, cysteine, beta-mercaptoethanol, E64, and elastatinal had little effect on enzyme activity. In addition, antiplasmin and antithrombin III were not sensitive to CPM-2. Based on the results of a fibrinolytic activity test, CPM-2 readily cleaved Aalpha- and Bbeta-chains of fibrinogen and fibrin, and gamma-chain of fibrinogen more slowly. The nonspecific action of the enzyme resulted in extensive hydrolysis, releasing a variety of fibrinopeptides of fibrinogen and fibrin. Polyclonal antibodies of CPM-2 were reactive to the native form of antigen. The ELISA was applied to detect quantities, in nanograms, of the antigen in CPM-2 protein.

Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide.

Previous study showed that some Gram-negative bacteria possess human blood group activity. Among them, Escherichia coli O86 has high blood group B activity and weak blood group A activity. This is due to the cell surface O-antigen structure, which resembles that of human blood group B antigen. In this study, we sequenced the entire E. coli O86 antigen gene cluster and identified all the genes responsible for O-antigen biosynthesis by sequence comparative analysis. The blood group B-like antigen in E. coli O86 O-polysaccharide was synthesized by sequentially employing three glycosyltransferases identified in the gene cluster. More importantly, we identified a new bacterial glycosyltransferase (WbnI) equivalent to human blood group transferase B (GTB). The enzyme substrate specificity and stepwise enzymatic synthesis of blood group B-like antigen revealed that the biosynthetic pathway of B antigen is essentially the same in E. coli O86 as in humans. This new finding provides a model to study the specificity and structure relationship of blood group transferases and supports the hypothesis of anti-blood group antibody production by bacterial stimulation.

Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone.

Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 was found to oxidize nitrobenzene (NB) to form m-nitrophenol (m-NP, 72%) and p-NP (28%) with an initial rate of 0.098 and 0.031 nmol/(min mg protein), respectively. It was also discovered that wild-type ToMO forms 4-nitrocatechol (4-NC) from m-NP and p-NP with an initial rate of 0.15 and 0.0082 nmol/(min mg protein), respectively, and 3-NC (12%) and nitrohydroquinone (NHQ, 88%) from o-NP with an initial rate of 0.11 and 0.8 nmol/(min mg protein), respectively. To increase the oxidation rate and alter the oxidation regiospecificity of nitro aromatics as well as to study the role of the active site residues I100, Q141, T201, and F205 of the alpha hydroxylase fragment of ToMO (TouA), DNA shuffling and saturation mutagenesis were used to generate random mutants. The mutants were initially identified by screening via a rapid agar plate assay and then were further examined by high-performance liquid chromatography (HPLC) and gas chromatography (GC). Several mutants with higher rates of activities and with different regiospecificities were identified; for example, Escherichia coli TG1 cells expressing either TouA mutant M180T/E284G or E214G/D312N/M399V produce 4-NC 4.5- and 20-fold faster than wild-type ToMO (0.037 and 0.16 nmol/min mg protein from p-NP, respectively). TouA mutant A107T/E214A had the regiospecificity of NB changed significantly from 28% to 79% p-NP. From 200 microM NB, TouA variants A101T/M114T, A110T/E392D, M180T/E284G, and E214G/D312N/M399V produce 4-NC whereas wild-type ToMO does not. From m-NP, TouA mutant I100Q produces 4-NC (37%) and NHQ (63%), whereas wild-type ToMO produces only 4-NC (100%). Variant A107T/E214A acts like a para enzyme and forms p-cresol as the major product (93%) from toluene with enhanced activity (2.3-fold), whereas wild-type ToMO forms 32%, 21%, and 47% of o-, m-, and p-cresol, respectively. Hence, the non-specific ToMO was converted into a regiospecific enzyme, which rivals toluene 4-monooxygenase of P. mendocina KR1 and toluene o-monooxygenase of Burkholderia cepacia G4 in its specificity.

Genotoxicity evaluation of Isaria sinclairii (ISE) extract.

The mutagenic potential Isaria sinclairii, a traditional Chinese medicine composed of the fruiting bodies of I. sinclairii and its parasitic host larva, was evaluated using short-term genotoxicity tests, namely, the Ames test, chromosome aberration (CA), and micronuclei (MN) tests. In a Salmonella typhimurium assay, I. sinclairii extract (ISE) did not produce any mutagenic response in the absence or presence of 59 mix with TA98, TA100, TA1535, and TA1537. In the chromosome aberration (CA) test, ISE induced no significant effect on Chinese hamster ovary (CHO) cells compared with control. In the MN test, no significant change in the occurrence of micronucleated polychromatic erythrocytes was observed in male ICR mice intraperitoneally administered ISE at doses of 15, 150, or 1500 mg/kg. These results indicate that ISE has no mutagenic potential in these in vitro and in vivo systems.

Photoactivation of pheophorbide a induces a mitochondrial-mediated apoptosis in Jurkat leukaemia cells.

The mechanism of cell death by pheophorbide a (Pba) which has been established to be a potential photosensitizer was examined in experimental photodynamic therapy (PDT) on Jurkat cells, a human lymphoid tumor cell line. In 30-60 min after irradiation, Pba treated cells exhibited apoptotic features including membrane blebbing and DNA fragmentation. Pba/PDT caused a rapid release of cytochrome c from mitochondria into the cytosol. Sequentially, activation of caspase-3 and the cleavage of poly ADP-ribose polymerase (PARP) were followed. Meanwhile, no evidence of activation of caspase-8 was indicated in the cells. In experiments with caspase inhibitors, it was found that caspase-3 alone was sufficient initiator for the Pba-induced apoptosis of the cells. Pba specific emission spectra were confirmed in the mitochondrial fraction and the light irradiation caused a rapid change in its membrane potential. Thus, mitochondria were entailed as the crucial targets for Pba as well as a responsible component for the cytochrome c release to initiate apoptotic pathways. Taken together, it was concluded that the mode of Jurkat cell death by Pba/PDT is an apoptosis, which is initiated by mitochondrial cytochrome c release and caspase-3-pathways.