Induced Expression of Cancer Stem Cell Markers ALDH1A3 and Sox-2 in Hierarchical Reconstitution of Apoptosis-resistant Human Breast Cancer Cells.

We established an experimental system that can induce p53-dependent apoptosis by doxycycline treatment to analyze characteristics of the apoptosis-resistant cancer cell subpopulation in the human breast cancer cell line HCC1937. Expression patterns of the stem cell markers, ALDH1A3 and Sox-2, the luminal differentiation marker, GATA3 and the proliferation index marker, Ki-67 were analyzed using immunostaining and fluorescence-activated cell sorting (FACS). After doxycycline treatment, the number of viable cells was gradually decreased over seven days in a time-dependent manner due to p53-induced apoptosis; however, the number of smaller-sized ALDH1A3+ cells assessed by immunostaining increased sharply after 1 day of doxycycline treatment, suggesting their apoptosis-resistant nature. The expression of ALDH1A3 was also detected in 78% of small-sized Ki-67+ proliferating progenitor cells, followed by the transient expression of GATA3, which presumably indicated the ability to differentiate into luminal progenitor cells. Although 42.2-58.5% of residual cells were positive for both ALDH1A3 and GATA3, their expression patterns exhibited an inverse correlation. The expression pattern of another stem cell marker, Sox-2, was similar, but more drastically altered after p53 induction compared with ALDH1A3. These findings may aid in understanding the hierarchical responses of cancer stem cells to therapeutic stresses.

Effects of Helicobacter pylori infection on gastric parietal cells and E-cadherin in Mongolian gerbils.

Atrophic gastritis caused by infection with Helicobacter pylori is characterized by parietal cell loss, which is a main risk factor for gastric cancer. Parietal cells play a crucial role in the regulation of cell lineage maturation and proliferation in the gastric units. Among the classical cadherins, E-cadherin plays an important role not only in epithelial cell-cell connections, but also in the maintenance of epithelial polarity and gastric glandular architecture and regulation of cell proliferation. The aim of this study is to elucidate how parietal cells and E-cadherin are altered in gastritis with Helicobacter pylori infection. We studied the effects of Helicobacter pylori on gastric mucosal E-cadherin 2 weeks after inoculation and investigated the relationship between parietal cell loss and the amount of E-cadherin on parietal cells in Mongolian gerbils. The number of parietal cells and amount of staining of E-cadherin below the isthmus were investigated by immunohistochemistry. It was shown that a reduction in intercellular E-cadherin preceded the disappearance of parietal cells. The gastric glands where parietal cells were lost were replaced by mucus secreting cells without E-cadherin. These results suggest that Helicobacter pylori damaged E-cadherin on parietal cells and caused massive parietal cell loss, leading to the deregulation of gastric morphology.

Photoinduced electron transfer in photorobust coumarins linked with electron donors affording long lifetimes of triplet charge-separated states.

Electron donor (D) substituted 3-ethoxycarbonylcoumarin (CM) derivatives [D-CM: D = 4-diphenylaminophenyl (DPA), 4-diethylaminophenyl (DEA), 4-dimethylaminophenyl (DMA), and 2-methyl-4-dimethylaminophenyl (MeDMA)] are synthesized and characterized. Photoinduced electron transfer (ET) from the D moiety to the acceptor (CM) and back electron transfer (BET) are investigated by femtosecond and nanosecond laser flash photolysis measurements. Femtosecond laser excitation at 355 nm of a deaerated acetonitrile (MeCN) solution of D-CM shows generation of the singlet charge-separated (CS) state [(1)(D(.+)-CM(.-))] by ET from D to the singlet excited state of the CM moiety ((1)CM*), and this is followed by rapid decay within 3 ns to afford the triplet excited state (D-(3)CM*). Nanosecond laser excitation of a deaerated MeCN solution of D-CM results in formation of the triplet CS state by ET from D to (3)CM*. The quantum yield of formation of the triplet CS state [(3)(DPA(.+)-CM(.-))] in the presence of iodobenzene (PhI) in deaerated MeCN increases with increasing concentration of PhI to reach 27 % at 0.5 M PhI. The triplet CS state decays by bimolecular BET because of the long CS lifetime by unimolecular BET. Formation of the long-lived triplet CS state was confirmed by electron spin resonance (ESR) measurements. The photorobust nature of DPA-CM is demonstrated by multiple laser pulse excitation (>1000 times) at 355 nm. The photoinduced ET and BET rate constants of a series of D-CM are thoroughly analyzed in light of the Marcus theory of electron transfer.

Inter- and intramolecular photoinduced electron transfer of flavin derivatives with extremely small reorganization energies.

Photoinduced electron transfer (ET) of a series of aromatic electron donors (D) to the singlet or triplet excited state of a flavin analogue (10-methylisoalloxazine: MeFl) and intermolecular back electron transfer (BET) from MeFl(*-) to D(*+) in benzonitrile (PhCN) has been investigated in light of the Marcus theory of ET. The rate constants of intermolecular photoinduced ET (k(et)) from D to the singlet excited state ((1)MeFl*) and the triplet excited state ((3)MeFl*) were determined by fluorescence quenching and enhanced decay rates of triplet-triplet (T-T) absorption by the presence of D, respectively. The k(et) values increase with an increase in the ET driving force to reach the diffusion-limit value that remains constant with a further increase in the ET driving force. Nanosecond laser flash photolysis was performed to determine the rate constants of intermolecular BET (k(bet)) from MeFl(*-) to D(*+) in PhCN. In contrast to the case of k(et), the driving force dependence of k(bet) shows a pronounced decrease towards the highly exothermic region. The reorganization energy (lambda) of intermolecular BET is determined to be 0.68 eV by applying the Marcus equation in the inverted region, where the k(bet) value decreases with increasing the BET driving force. The slowest BET was observed for BET from MeFl(*-) to N,N-dimethylaniline radical cation (DMA(*+)) with the k(bet) value of 3.5 x 10(6) M(-1) s(-1), which is 1600 times smaller than the diffusion rate constant in PhCN (5.6 x 10(9) M(-1) s(-1)). Then, DMA was linked to the 10-position of isoalloxazine to synthesize a DMA-flavin linked dyad (10-[4'-(N,N-dimethylamino)phenyl]-isoalloxazine: DMA-Fl). Photoexcitation of DMA-Fl results in photoinduced ET from the DMA moiety to the singlet excited state of Fl moiety to form the charge-separated (CS) state (DMA(*+)-Fl(*-)) that has an extremely long lifetime (2.1 ms) in PhCN at 298 K.

Roles of NADPH oxidase in occurrence of gastric damage and expression of cyclooxygenase-2 during ischemia/reperfusion in rat stomachs.

NADPH oxidase is an enzyme that converts molecular oxygen into reactive oxygen species, which cause severe damage in several organs. Cyclooxygenase (COX)-2 is an inducible enzyme that is important in gastric mucosal defense and repair processes. It is unclear whether NADPH oxidase is related to COX expression in the gastric mucosa, so we investigated the correlation. Under urethane anesthesia, a male Sprague Dawley rat stomach was mounted in an ex-vivo chamber, and ischemia/reperfusion (I/R) was performed through a cannula in the femoral vein. I/R significantly increased NADPH oxidase activity, H(2)O(2) production, and myeloperoxidase (MPO) activity. In contrast, ischemia alone clearly enhanced both NADPH oxidase activity and H(2)O(2) production but not MPO activity. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) suppressed I/R-induced mucosal damage. On the other hand, the selective COX-2 inhibitor rofecoxib exhibited a tendency to enhance the severity of gastric damage induced by I/R, although the selective COX-1 inhibitor SC-560 and the nonselective COX inhibitor indomethacin had no effect. I/R also increased the expression of COX-2, and this increase was suppressed by pretreatment with DPI. These findings suggest that the increase in NADPH oxidase activity is involved in the occurrence of gastric mucosal damage induced by I/R and that this enzyme activity may be causally related to the upregulation of COX-2 during I/R.

Does bimolecular charge recombination in highly exergonic electron transfer afford the triplet excited state or the ground state of a photosensitizer?

The investigations were made on photoinduced electron transfer (ET) from the singlet excited state of rubrene (1RU*) to p-benzoquinone derivatives (duroquinone, 2,5-dimethyl-p-benzoquinone, p-benzoquinone, 2,5-dichloro-p-benzoquinone, and p-chloranil) in benzonitrile (PhCN) by using the steady state and time-resolved spectroscopies. The photoinduced ET produces solvent-separated type charge-separated (CS) species and the charge-recombination (CR) process between RU radical cation and semiquinone radical anions obeys second-order kinetics. Not only the CS species but also the triplet excited state of RU (3RU*) is seen in the transient absorption spectra upon laser excitation of a PhCN solution of RU and p-benzoquinone derivatives. The comparison of their time profiles clearly suggests that the CR process between RU radical cation and semiquinone radical anions to the ground state is independent from the deactivation of 3RU*. This indicates that the CR in a highly exergonic ET occurs at a longer distance with a large solvent reorganization energy, which results in faster ET to the ground state than to the triplet excited state that is lower in energy than the CS state. Photoinduced ET from 3RU* in addition from 1RU* also occurs when p-benzoquinone derivatives with electron-withdrawing substituents were employed as electron acceptors.

Cerebral correlates of the progression rate of the cognitive decline in probable Alzheimer's disease.

OBJECTIVE: To evaluate the possible relation between the rate of cognitive deterioration in patients with probable Alzheimer's disease (AD) and the distribution pattern of neural dysfunction. METHODS: The regional cerebral blood flow (rCBF) was measured in rapidly and slowly progressing groups of AD patients using single-photon emission computed tomography and was compared between the groups. While controlling for demographic and clinical factors that could be associated with the stage and prognosis of the illness, the deterioration rate of the Mini Mental State Examination (MMSE) score was significantly greater in the rapidly progressing group than that in the slowly progressing group. RESULTS: The rCBF in the right posterodorsal, anterior and superior prefrontal cortices and the inferior parietal cortex was significantly lower in the rapidly progressing patients. Moreover, lower perfusion in these regions correlated significantly with rapid deterioration in the MMSE. CONCLUSIONS: These findings suggest that the rCBF values in these cortical regions could be useful in predicting which AD patients will show a relatively rapid cognitive decline.

Regional cerebral blood flow abnormalities in nondemented patients with memory impairment.

BACKGROUND: Patients with objective evidence of memory impairment have been considered to be at risk for developing Alzheimer's disease (AD). However, little is known about patterns of regional cerebral blood flow abnormalities and their prognostic significance in these patients. METHODS: The authors retrospectively studied 28 nondemented subjects with memory loss and investigated patterns of blood flow abnormalities on single photon emission computed tomography (SPECT). RESULTS: The patients were followed up for more than 2 years; during follow-up, 14 patients (50%) developed AD. The onset of memory impairment in patients who progressed to AD was significantly earlier than in those who remained in a nondemented condition. SPECT data from the initial evaluation were analyzed by region of interest analysis and statistical parametric mapping. Interestingly, both groups of patients shared hypoperfusion in the medial temporal regions and the posterior cingulate. In addition to these regions, significant blood flow reduction in the parietal and anterior cingulate cortices was detected in patients who progressed to AD. CONCLUSIONS: These results demonstrate that (1) subjects with an earlier onset of memory loss have an increased risk for developing AD, (2) SPECT can be useful for distinguishing subjects with memory loss who will rapidly progress to AD from those who will not, and (3) perfusion impairment typical of AD was evident even in subjects with memory impairment who remained nondemented.