Tumour formation by single fibroblast growth factor receptor 3-positive rhabdomyosarcoma-initiating cells.

BACKGROUND: The hypothesis that malignant tumours are generated by rare populations of cancer stem cells that are more tumourigenic than other cancer cells has gained increasing credence. The objective of this study was to identify and characterise a subpopulation of human sarcoma-initiating cells. METHODS: We examined established rhabdomyosarcoma cell lines by flow cytometry. Tumourigenesis was examined by xenograft models. Real-time PCR and immunohistochemistry were performed to examine the gene expression using cell lines and biopsy specimens. RESULTS: Rhabdomyosarcoma cell lines included small populations of fibroblast growth factor receptor 3 (FGFR3)-positive cells. FGFR3-positive KYM-1 and RD cells were more strongly tumourigenic than FGFR3-negative cells. In addition, xenoengraftment of 33% of single FGFR3-positive KYM-1 cells yielded tumour formation. Stem cell properties of FGFR3-positive cells were further established by real-time PCR, which demonstrated upregulation of undifferentiated cell markers and downregulation of differentiation markers. We showed that in the absence of serum, addition of basic fibroblast growth factor maintained and enriched FGFR3-positive cells. On the other hand, ciliary neurotrophic factor reduced the proportion of FGFR3-positive cells. Real-time PCR and immunohistochemical examination revealed that embryonal rhabdomyosarcoma patient biopsy specimens were found to over-express FGFR3. CONCLUSIONS: Our findings suggest that rhabdomyosarcoma cell lines include a minor subpopulation of FGFR3-positive sarcoma-initiating cells, which can be maintained indefinitely in culture and which is crucial for their malignancy.

The olfactory bulb: coding and processing of odor molecule information.

Olfactory sensory neurons detect a large variety of odor molecules and send information through their axons to the olfactory bulb, the first site for the processing of olfactory information in the brain. The axonal connection is precisely organized so that signals from 1000 different types of odorant receptors are sorted out in 1800 glomeruli in the mouse olfactory bulb. Individual glomerular modules presumably represent a single type of receptor and are thus tuned to specific molecular features of odorants. Local neuronal circuits in the bulb mediate lateral inhibition among glomerular modules to sharpen the tuning specificity of output neurons. They also mediate synchronized oscillatory discharges among specific combinations of output neurons and may contribute to the integration of signals from distinct odorant receptors in the olfactory cortex.

Kumanasamuha geaster sp. nov., an anamorph of Chorioactis geaster from Japan.

A new species of Kumanasamuha is described and illustrated from axenic single-spore isolates of Chorioactis geaster. The characteristics of conidia and hyphae are the same as the dematiaceous hyphomycete observed on decayed trunks of Quercus spp., where C. geaster appeared in the field in Japan. Our results indicated that C. geaster in situ colonizes decayed trunks of Quercus spp.

Triply bridged dinuclear ruthenium complexes bearing alkylbis(2-pyridylmethyl)amine in the mixed-valence state of Ru(ii)-Ru(iii).

Triply halogeno and methoxido-bridged dinuclear ruthenium complexes bearing a tridentate ancillary ligand, alkylbis(2-pyridylmethyl)amine (alkyl, ethyl and benzyl), in the Ru(ii)-Ru(iii) mixed-valence state were synthesized by reduction reactions of the trichloridoruthenium(iii) complex, fac-[RuIIICl3(ebpma)], followed by chlorido-substitution and oxidation reactions in air. The conversion of the bridging ligands of the diruthenium complexes was also made possible through reduction of the dinuclear core. The electronic structures of the mixed-valence state were investigated by electron spin resonance (ESR), X-ray crystallography, electrochemical measurements and UV-vis-near infrared (NIR) spectroscopy. The mixed-valence state of all the triply bridged complexes was stable and classified as Class III.

Oxygen at high pressures: a theoretical approach to monoatomic phases.

We have studied the ζ-phase of solid oxygen using the generalized gradient approximation in the density functional approach. Calculations of total energies and pressures have been carried out for the prototype of diatomic ζ-phase and other hypothetical monoatomic crystal structures. The diatomic phase was found to be stable over a wide range of pressure (100-2000 GPa). The stacking of molecular layers is discussed in comparison with the available experimental data.

Assessment of relevant fungal species in clinical solid wastes.

The study aimed to determine the fungal diversity in clinical waste samples from a healthcare facility in Penang Malaysia. Different fungi species were detected in 83.75 % of the 92 clinical waste samples that were screened from different sections of the healthcare facility. One hundred fifty fungal isolates comprising of 8 genera and 36 species were obtained. They were purified by using single spore isolation technique. Subsequently, the isolates were identified by phenotypic method based on morphological and culture characteristics on different culture media. Among all fungal isolates, Aspergillus spp. in section Nigri 10.2 %, Aspergillus niger 9.5 %, Aspergillus fumigatus 8.8 %, Penicillium. simplicissium 8 %, Aspergillus tubingensis 7.3 %, Aspergillus terreus var. terreus 6.6 %, Penicillium waksmanii 5.9 % and Curvularia lunata 6.5 % were the most frequent. Among five sections of the Wellness Centre, the clinical wastes collected from the diagnostic labs of haematology section had the highest numbers of fungal species (29 species). Glove wastes had the highest numbers of fungal species (19 species) among 17 types of clinical wastes screened. Among all fungal species, Aspergillus spp. exhibited higher growth at 37 °C than at 28 °C, indicating the potential of these opportunistic fungi to cause diseases in human. These results indicated the potential of hospital wastes as reservoirs for fungal species.

Tea catechins decrease micellar solubility and intestinal absorption of cholesterol in rats.

A(-)-epicatechin (EC) and (-)-epigallocatechin (EGC) mixture and a mixture of their gallates (ECG and EGCG, respectively) markedly lowered lymphatic cholesterol absorption in rats with a cannulated thoracic duct. A mixture of ECG and EGCG was more effective in reducing cholesterol absorption than the EC and EGC mixture. These catechins also tended to decrease lymphatic absorption of triacylglycerols, although not so pronounced as in cholesterol absorption. An in vitro study on micellar solubility of cholesterol showed that these catechin mixtures precipitated cholesterol solubilized in mixed bile salt micelles in a dose-dependent manner. A mixture of ECG and EGCG more effectively precipitated micellar cholesterol than a mixture of EC and EGC. When purified EC, EGC, ECG and EGCG were used, EGCG was more effective in precipitating micellar cholesterol than ECG. The effect of EC and EGC was comparable and weaker than their gallate esters. The bile acid concentration in the micelles was not affected by these catechins. A positive correlation was observed between the amount of coprecipitated EGCG and cholesterol. These results clearly show that tea catechins, in particular their gallate esters, effectively reduce cholesterol absorption from the intestine by reducing solubility of cholesterol in mixed micelles. The observation accounts for the hypocholesterolemic effect of tea catechins.

Effects of adenylyl cyclase-linked neuropeptides on the expression of ciliary neurotrophic factor-mRNA in cultured astrocytes.

Ciliary neurotrophic factor (CNTF) is a molecule which has profound effects on various neural cell types. In the central nervous system, expression of CNTF-mRNA is highly concentrated in olfactory bulb. In the present study, we examined the regulatory mechanism of CNTF-mRNA expression in cultured astrocytes from newborn rat brain. Cultured astrocytes from new born rat brain expressed CNTF-mRNA at levels comparable to the level in olfactory bulb in vivo. Treatment of the astrocytes with forskolin, an activator of adenylyl cyclase, led to a decrease of CNTF-mRNA level. The effect of forskolin was mimicked by cAMP-linked agonists, such as VIP, PACAP, isoproterenol and dopamine. Cycloheximide, an inhibitor of protein synthesis, did not abolish the forskolin-induced decrease of CNTF-mRNA. Measurement of the half-life of CNTF-mRNA in the presence of actinomycin D, an inhibitor of transcription, indicated that the degradation of CNTF-mRNA is not destabilized by the forskolin-treatment. These data taken together suggest that the cAMP-induced suppression of CNTF-mRNA is mainly caused by the inhibition of CNTF gene transcription.

Developing germ cells in mouse testis express pheromone receptors.

Pheromone receptors are expressed in the accessory olfactory system, which is vital for non-specific chemical communication and for sexual behavior. Under the hypothesis that some of the pheromone molecules released from female reproductive organs might regulate sperm chemotaxis or chemokinesis, we examined whether the V1R type pheromone receptor mRNAs are expressed in developing germ cells. By a reverse transcription-PCR method, we obtained nine kinds of cDNA fragments belonging to the receptor family. In situ hybridization analysis in testicular sections using probes of testicular pheromone receptors (TVRs) revealed that TVR mRNAs were expressed by spermatids. TVRs were also expressed in the accessory olfactory organ. In the testis, hybridization signals were localized in subsets of the seminiferous tubules, suggesting that TVRs were expressed by selective subsets of the spermatids. In situ hybridization study suggests also that each sperm expresses multiple pheromone receptors. The testicular pheromone receptors might have an important role in the maturation and/or migration of sperm.

Remarkable activity enhancement of thermolysin mutants.

Most attempts to modify the properties of enzymes by amino acid substitution around the active sites have resulted in suppression of the biological activity, suggesting that the structure of natural enzymes should be almost optimized evolutionally to show the highest activity. In contrast, we found an interesting site of a well-known metalloendopeptidase, thermolysin (EC.3.4.24.4), where almost all the amino acid replacement causes a remarkable increase in the hydrolytic activity. Negative correlation between the activity and the thermal stability was observed. The flexibility around the substrate binding site is suggested to be a key to the correlation. Nature may have selected the amino acid at this site, which suppresses the flexibility of the molecule, to get the highest thermal stability at the expense of the activity.

Two mirror-image sensory maps with domain organization in the mouse main olfactory bulb.

The glomerular sheet in the olfactory bulb (OB) provides an olfactory sensory map identifying which odorant receptors (ORs) in the nose are activated by inhaled odorants. How are the glomeruli spatially arranged in the OB? Using OCAM and neuropilin-1 (NP1) as molecular markers for target glomeruli of distinct subsets of olfactory axons, we demonstrate here that glomeruli are parceled into topographically distinct domains. Spatial arrangement of these domains suggests that each OB contains two mirror-image maps of the glomeruli. In situ hybridization shows that the glomeruli representing the same OR are symmetrically arranged; one in a domain in the lateral hemisphere and the other in a corresponding domain in the medial hemisphere of the OB. These results suggest that OB contains two symmetrical OR maps with similar domain organization.

Repression of nitrogenase by ethanol in nitrogen-deprived cultures of Rhodovulum sulfidophilum.

Light-dependent H2 evolution did not occur in nitrogen-deprived cultures of Rhodovulum sulfidophilum in the presence of ethanol. When ethanol was added to cells which had been grown with ammonia, derepression of the nitrogen fixation genes (nifHD) was inhibited at an ethanol concentration of 1 mM. On the other hand, when cells had nitrogenase-catalyzed proton-reducing activity prior to ethanol addition, reduction of the nifHD transcript level did not occur after the addition. In cells grown with ammonia, concomitant addition of an auxiliary oxidant such as dimethylsulfoxide or sodium bicarbonate resulted in derepression of nitrogenase activity in the presence of ethanol. These results suggest that the electron-accepting process is necessary for derepression of nif genes in cultures which use ethanol as the electron donor.

Effects of phorbol ester on expression of CNTF-mRNA in cultured astrocytes from rat olfactory bulb.

Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine which has various functions, such as survival promoting effect on both peripheral and central neurons, promotion of cholinergic differentiation, and participation in differentiation of Type-2 astrocytes (reviewed in ref. [30]). However, the regulatory mechanism of the CNTF expression is largely unknown. In this study, we analyzed the effects of phorbol 12-myristate 13-acetate (PMA), an activator of PKC, on the expression of CNTF-mRNA in cultured astrocytes from neonatal rat olfactory bulb. PMA induced a transient decrease of CNTF-mRNA levels which was followed by a persistent increase of the mRNA up to 4-fold of the control level at 24 h after the addition of the compound. Both the PMA-induced decrease and increase of the CNTF-mRNA levels were canceled by treatment with cycloheximide, an inhibitor of protein synthesis, suggesting that protein synthesis-dependent mechanisms participate in both the PMA-induced decrease and increase of CNTF-mRNA levels. On the other hand, PMA induced expressions of mRNAs of several subunit members of the AP-1 complex, such as c-fos, c-jun and jun-B. Furthermore, dexamethasone, a synthetic glucocorticoid which is known to inhibit the AP-1 complex-mediated transcription [14,27,36], canceled the PMA-induced decrease of the CNTF-mRNA levels. These results suggested that the AP-1 complex participates in the regulatory mechanism of the CNTF expression in the cultured astrocytes treated with PMA.

The effect of GABAergic system activity on hyperthermia-induced seizures in rats.

Developing rats were given GABA antagonists and agonist before electrical seizure discharges were induced by heating the brain of a rat with infra-red rays. The thresholds for the GABA antagonist groups were significantly lower than that for the control, and the threshold for the GABA agonist group was significantly higher than that for the control. These results support the hypothesis that reduced GABAergic system activity underlies febrile seizures.

The influence of blood gas changes on hyperthermia-induced seizures in developing rats.

Fever induces seizures in infants with febrile convulsions or epilepsy. Hyperpnea induced by fever may contribute to the induction of these seizures. In order to examine this possibility, we evaluated the effect of changes in arterial blood gas tension on hyperthermia-induced seizures in developing rats. Electrical seizure discharges were induced by application of infra-red rays on the skull of rats under mechanical ventilation with different respiratory conditions. There was positive correlation between pCO(2) and the seizure threshold (ST) defined as a latency from the start of hyperthermia to the occurrence of seizures: ST (seconds, s) = 2.36 pCO(2) + 0.05 (R(2) = 0.80, P < 0.001). Seizure duration (SD) was longer at lower pCO(2) level: 18 (6-33) (median, range) s at pCO(2) ranging from 23 to 26 mmHg vs. 0 (0-7) s at pCO(2) ranging from 35 to 57 mmHg (P < 0.01). Hypoxia significantly increased ST: 84 (61-100) s at P0(2) ranging from 53 to 76 mmHg vs. 60 (51-72) s at P0(2) ranging from 87 to 131 mmHg (P < 0.01). Hyperoxia prolonged SD: 27 (10-30) s at P02 ranging from 100 to 170 mmHg vs. 9 (0-23) at P0(2) ranging from 53 to 93 mmHg (P < 0.02). Hypocarbia caused by fever-induced hyperpnea probably contributes to the generation of fever-induced seizures.

The pathogenic role of the NMDA receptor in hyperthermia-induced seizures in developing rats.

Hyperthermia-induced seizures (HS) in rats have been used as a model of febrile seizures. Activation of the N-methyl-D-aspartate (NMDA) receptor by increased extracellular glutamate (Glu) in the cortex during hyperthermia may be involved in the induction of HS and HS kindling. To confirm this hypothesis, the effects of a potent blocker of the NMDA receptor, MK-801, on the threshold and pattern of HS were evaluated. The threshold temperatures for rats given 0.1 (low dose) and 0.5 (high dose) mg/kg MK-801 (i.p.) for the first time were 41.6 degrees C (39.7-42) (median, range) and 42.0 (41.2-42.0), respectively, which were significantly higher than the 40.5 (39.4-41.2) for rats without MK-801 administration (P < 0.01). The recurrent occurrence of HS suppressed the increase in the threshold temperature with age, and changed the seizure from partial to generalized seizures (HS kindling), whereas these effects of recurrent HS on the threshold and pattern of HS were inhibited by the high dose (0.5 mg/kg) of MK-801. MK-801 blocks HS and HS kindling. The activation of the NMDA receptor during hyperthermia plays an important role in the induction of HS and HS kindling.

Lymphatic transport of eicosapentaenoic and docosahexaenoic acids as triglyceride, ethyl ester and free acid, and their effect on cholesterol transport in rats.

Lymphatic transport of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids given in the forms of triglyceride, ethyl ester of free acid and their effect on cholesterol transport was compared in lymph-cannulated rats. Lymphatic recovery of DHA and EPA given by stomach tube in the form of triglyceride in which they were mainly located at the 2-position was significantly higher than that of the ethyl ester or free acid during the first 6 hr after the administration and the tendency continued until 9 hr. In contrast, the 9 to 24 hr recovery of DHA and EPA in the forms of ethyl ester and free acid was considerably higher than that of triglyceride. Consequently, cumulative 24 hr recovery of EPA was comparable among the three forms. However, the 24 hr recovery of DHA was highest in free acid, lowest in ethyl ester and intermediate in triglyceride. Recovery of the free acid between 9 and 24 hr after administration was significantly higher than that given in the forms of triglyceride or ethyl ester. Cholesterol recovery in lymph of rats given with ethyl ester or free acid was lower than that given with triglyceride at an early stage after the administration in both EPA and DHA. Cumulative 24 hr recovery of cholesterol in rats given these fatty acids as ethyl ester was significantly lower than in those given as the other two forms.

Clinical study of epilepsy with severe febrile seizures and seizures induced by hot water bath.

We followed five cases with severe febrile seizures (FS) with early onset and seizures induced by hot water bath, indicating severe myoclonic epilepsy in infancy (SME) or the peripheral form of SME. These cases, however, had far better clinical courses than that of SME or the peripheral form of SME. These cases were characterized by: (a) severe FS with early onset and seizures induced by hot water bath; (b) a lack of myoclonic seizures; (c) no psychomotor deterioration; (d) no underlying disorders or signs of brain insult on neurologic and laboratory examination; (e) relatively successful seizure control; (f) none of the severe EEG abnormalities seen in SME and the peripheral form of SME; and (g) a positive family history of convulsive disorders.

Long-term medical, educational, and social prognoses of childhood-onset epilepsy: a population-based study in a rural district of Japan.

The long-term prognosis of childhood-onset epilepsy has rarely been studied in a general population. We examined the long-term medical, educational, and social outcomes in individuals with a history of childhood-onset epilepsy aged 20 years or older in a defined area of Japan. Furthermore, the patients' recognition of epilepsy as well as the parents' concerns about the prognosis of epilepsy were also surveyed. After a mean follow-up period of 18.9 years, we had sufficient data on 148 surviving patients aged 20-38 years (mean, 26. 0 years) and seven dead patients. The follow-up rate was 92.8%. In the overall group, the percentages of those who had had each of the following were as follows: (a) 5-year remission, 62.8%; (b) a relapse of seizures, 17.4%; (c) psychiatric complications, 2.7%; (d) mortality, 4.5%; (e) attendance at regular classes of an ordinary school during compulsory education, 71.6%; (f) entrance to high school, 65.5%; (g) employment, 67.4%; (h) marriage, 23.0%; and (i) acquisition of a driver's license, 54.7%. The educational and social variables of the control population were as follows: (e) 99.1, (f) 97.0, (g) 96.6, (h) 51.9, and (i) 94.8%. In the 99 patients of normal intelligence, the results of the same analysis were as follows: (a) 75.8, (b) 10.7, (c) 0, (d) 0.6, (e) 100, (f) 96.0, (g) 95.2, (h) 33.3, and (i) 77.8%, in contrast to the corresponding variables of the 49 patients with mental retardation, that is, (a) 36.7, (b) 44.4, (c) 8.2, (d) 12.2, (e) 14.3, (f) 6.1, (g) 20.4, (h) 2.0, and (i) 4.1%, respectively. The best predictors of seizure remission included an early response to therapy, a low frequency of seizures or an absence of status epilepticus prior to therapy, and normal mental development. As for the current awareness of epilepsy and its prognosis, nearly 40% of the patients did not know the true name of their illness, and the same proportion of parents were still anxious about the prognosis even if their children had been taken off medication. Our data show that the majority of patients with childhood-onset epilepsy have a favorable long-term medical prognosis in terms of seizure remission and psychiatric complications, whereas they have a higher mortality rate and lower levels of educational background as well as employment and marital status compared with the general population. However, except for the low marriage rate for the younger age group and the low rate of holding a driver's license, patients of normal intelligence appear to have more favorable long-term prognoses in terms of educational and social aspects as well. In Japan, individuals with epilepsy may not seek a driver's license because of the law.