Saturated fatty acid induces cancer stem cell-like properties in human hepatoma cells.

Hepatic steatosis has been reported to be a risk factor for the development of liver cancer. The underlying mechanism for carcinogenesis remains to be elucidated. It has been postulated that cancer stem cells (CSCs) within tumor tissues are a subset of cells with stem cell properties of self-renewal and undifferentiation. The purpose of this study was to investigate the effects of a saturated fatty acid, palmitate (PA), on CSC-like properties of human hepatoma HepG2 cells. We investigated the effects of PA on HepG2 cells and primary rat hepatocytes (PRH) by exposing them to PA to induce lipid accumulation. Significant fat accumulation was observed by Oil Red O staining in cells exposed to PA, and it was accompanied by significant increase in NFκB (p65) nuclear translocation in HepG2 cells. Notably, PA significantly enhanced the sphere forming ability of HepG2 cells, but not PRH. Furthermore, PA significantly increased stemness gene expressions of Sox2 and Oct4, and sonic hedgehog (Shh) production. Notably, NFκB inhibitors, N-Acetyl-L-cysteine and pyrollidine dithiocarbamate, and a NOX inhibitor, diphenyleneiodonium, significantly attenuated PA-induced sphere forming ability of HepG2 cells. Our results suggest that lipid accumulation may not only induce pro-inflammatory responses in hepatocytes but may also activate CSC-like properties of hepatoma cells through NFκB activation.

Characteristics of patients with Clostridium difficile infection in Taiwan.

The medical records of 84 patients with stool cultures positive for Clostridium difficile during the period August 2007 to June 2009 were retrospectively reviewed. A case of confirmed (toxigenic)C. difficile infection (CDI) was defined by the presence of symptoms (fever, diarrhoea, abdominal discomfort or distension, ileus) and the presence of toxigenic C. difficile. Patients with compatible clinical symptoms and stool cultures positive for non-toxigenic C. difficile isolates were defined as probable (non-toxigenic) CDI cases. Of these 84 patients, 50 (59.5%) were diagnosed as confirmed CDI and 34 (40.5%) as probable CDI. Thirteen (15.5%) of the 84 patients died during their hospital stay. Usage of proton pump inhibitors was a significant independent risk factor for CDI (OR 3.21, P=0.014). Of the 50 isolates associated with confirmed CDI, seven (8.3%) carried binary toxin genes (cdtAB), and six (7.1%) had a deletion in the tcdC gene. The mortality rate in confirmed CDI patients with isolates exhibiting deletion in the tcdC gene (2/6, 33.3%), those with isolates harbouring binary toxin genes (2/7, 28.6%), and those with isolates containing mutations in gyrA (2/7, 28.6%) and gyrB (1/2, 50%) was higher than the overall mortality rate (10/50, 20%) in patients with confirmed CDI.

Sacrococcygeal teratoma in adults: case report and literature review.

Sacrococcygeal teratoma is one of the most common tumours in infants but rare in adults. We present a case of sacrococcygeal teratoma in a female adult. The clinical presentation, radiological and histological findings, management, and outcome are described.

Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents.

We encountered beta-mercaptoethanol-dependent artifact signals in western blot analyses using polyclonal antisera. Replacing beta-mercaptoethanol with dithiothreitol in the loading buffer did not eliminate the artifact signals. However, lowering the concentration of either dithiothreitol or beta-mercaptoethanol eliminated the background problems and allowed specific detection of the target protein. These results are consistent with the background signal being caused by anti-keratin antibodies in the antisera and keratin contamination of reagents. This study highlights the importance of testing a range of reducing agent concentrations when trying to eliminate artifact bands from western blots. However, this method may not be applicable when target proteins have disulfide bridges.

Effects of different durations of sustained inflation during cardiopulmonary resuscitation on return of spontaneous circulation and hemodynamic recovery in severely asphyxiated piglets.

OBJECTIVE: We previously demonstrated that sustained inflation (SI) during chest compression (CC) significantly reduces time to return of spontaneous circulation (ROSC) when compared to 3:1 compression:ventilation (C:V) ratio during neonatal resuscitation. However, the optimal length of SI during CC to improve ROSC and hemodynamic recovery in severely asphyxiated piglets is unknown. AIM: To examine if different lengths of SI will improve ROSC and hemodynamic recovery in severely asphyxiated piglets. INTERVENTION AND MEASUREMENTS: Thirty newborn piglets (1-3 days) were anesthetized, intubated, instrumented and exposed to 30-min normocapnic hypoxia followed by asphyxia. Piglets were randomized into four groups: 3:1 C:V (n = 8), CC with an SI duration of either 20 s (CC+SI 20) (n = 8) or 60 s (CC+SI 60) (n = 8), and a sham group (n = 6). Cardiac function, carotid blood flow, cerebral and renal oxygenation as well as respiratory parameters were continuously recorded throughout the experiment. MAIN RESULTS: When compared with 3:1 group, both CC+SI 20 and CC+SI 60 groups had significantly shorter ROSC time (p = 0.002). All three intervention groups had similar hemodynamic recovery by the end of 4 h observation period. There was no difference in lung injury markers among all experimental groups. However, when compared to the sham group, the concentrations of IL-6 (thalamus) and IL-6 + IL-8 (frontoparietal cortex) of the 3:1 C:V group were significantly higher, respectively. CONCLUSIONS: Even though relatively less animals achieved ROSC, CC during SI significantly improved ROSC time compared to 3:1 C:V in asphyxiated newborn piglets. However, there was no difference in ROSC characteristics and hemodynamic recovery between two CC+SI groups.

Tidal volume delivery during continuous chest compressions and sustained inflation.

OBJECTIVE: To determine the distending pressure needed to achieve sufficient tidal volume (VT) delivery during continuous chest compressions (CC) superimposed by sustained inflation (SI) (CC+SI). DESIGN: Randomised animal/manikin trial. SETTING: University laboratory. SUBJECTS: Cadaver piglets/manikin. INTERVENTIONS: SI distending pressures of 5, 10, 15, 20, 25 and 30 cm H2O were delivered in random order during CC+SI for 2 min each. MAIN OUTCOME MEASURES: VT, gas flow and airway pressure. Spearman's r for distending pressure and VT. RESULTS: Distending pressure and VT correlated in cadaver piglets (r=0.83, p<0.001), manikin (r=0.98, p<0.001) and combined data (r=0.49, p<0.001). VT was delivered during chest recoil during CC in both models. In cadaver piglets, a distending pressure ∼25 cm H2O was needed to achieve an adequate VT. CONCLUSIONS: Chest recoil generates VT depending on an adequate distending pressure. This has previously been demonstrated in adult animals. A pressure of ∼25 cm H2O is needed to achieve an adequate VT delivery.

Dopamine and thermoregulation: an evaluation with special reference to dopaminergic pathways.

The complex role of dopamine (DA) in the diencephalic mechanisms involved in the control of body temperature is reviewed and evaluated. In the context of the monoamine theory of thermoregulation, catecholaminergic synapses in the anterior hypothalamic pre-optic area, are proposed mediate the pathways in the brain-stem which subserve heat dissipation. Within this theoretical framework, hypothalamic DA is considered to underlie a portion of the functional component of the heat loss system. This deduction is based on pharmacological studies in which both the catecholamine and receptor antagonists have been infused directly into the hypothalamus. In view of the action of DA applied to the substantia nigra and other subcortical structures, the unique anatomical circuitry of the central dopaminergic projections has also been analyzed in terms of specific connections within critical morphological regions related to thermal functions. In particular, the nigro-striatal pathway could be involved in the mediation of one or more of the different aspects of the thermoregulatory system integrating both autonomic and behavioral responses. Finally, an anatomical schema which portrays the suggested mechanisms of DA activity is presented.

Calcitriol enhancement of TPA-induced tumorigenic transformation is mediated through vitamin D receptor-dependent and -independent pathways.

We previously showed that 1alpha,25-dihydroxyvitamin D3, calcitriol, enhanced phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced tumorigenic transformation of mouse epidermal JB6 Cl41.5a cells. To determine if calcitriol regulates this enhancement through a nuclear vitamin D receptor (VDR)-dependent or -independent pathway, we used vitamin D analogs which induce biological responses by either of these mechanisms. In JB6 Cl41.5a cells, 1alpha,24-dihydroxy-22-ene-24-cyclopropyl-vitamin D3 (BT), which like calcitriol binds to VDR and regulates transcription, inhibited cell growth, stimulated expression of nonphosphorylated osteopontin (OPN), and enhanced TPA-induced anchorage-independent growth (AIG, an in vitro assay which highly correlates with tumorigenicity of these cells). 25-Hydroxy-16-ene-23-yne-vitamin D3 (AT), which stimulates calcium influx but has low affinity for VDR, had moderate effects on cell growth and expression of OPN. However, it enhanced TPA-induced tumorigenic transformation, though to a lesser extent than BT, thus suggesting that a VDR-independent mechanism is involved. Since 1alpha-hydroxylase activity was detected in JB6 cells, AT could be converted into 1alpha,25-dihydroxy-16-ene-23-yne-vitamin D3 (V), an analog which binds with high affinity to VDR, and could subsequently enhance TPA-induced AIG. To verify whether the VDR-independent pathway is involved in calcitriol enhancement of tumorigenic transformation, two additional VDR-independent analogs, 1alpha,25-dihydroxy-lumisterol3 (JN) and 24R,25-dihydroxyvitamin D3 (AS), were tested. The analog JN, which stimulates calcium transport and cannot be further hydroxylated at 1-carbon position, increased TPA-induced AIG, while AS, which inhibits calcium influx, did not. These studies suggest that a VDR-independent pathway, perhaps stimulation of calcium influx, and a VDR-dependent mechanism, which directly affects transcription, are involved in calcitriol's enhancement of TPA-induced tumorigenic transformation in JB6 Cl41.5a cells.

Neurotensin perfusion of rat hypothalamus: dissociation of dopamine release from body temperature change.

To determine whether endogenous dopamine is involved in the impairment of body temperature induced by neurotensin, the local activity of [14C]dopamine in the hypothalamus of the unanesthetized rat was examined. A push-pull guide tube was implanted permanently above an intended site of perfusion within the anterior hypothalamic, pre-optic area or other region of the diencephalon. After the endogenous stores of dopamine at a specific site were labelled by microinjection of 0.02-0.05 muCi of [14C]dopamine, an artificial cerebrospinal fluid was perfused at the site at a rate of 20 microliter/min and at successive 5 min intervals. Perfusion of neurotensin in concentrations of 0.05 or 0.1 microgram/microliter in the dorsomedial hypothalamus, lateral hypothalamus, arcuate nucleus or diagonal band of Broca evoked a calcium-dependent efflux of [14C]dopamine. The release of dopamine induced by neurotensin was functionally specific since it was: (1) not mimicked by the relatively inactive neurotensin analogue, [D-Arg9]neurotensin; (2) dependent on the morphological locus of the push-pull perfusion; and (3) not accompanied by an efflux of [3H]norepinephrine when the site was double-labelled. Although neurotensin perfused in the anterior hypothalamic, pre-optic area caused a consistent decline in temperature, in most cases the temperature change did not correlate with an enhanced release of dopamine. Moreover, the release of dopamine, but not the temperature change, was abolished when neurotensin was perfused in a calcium-free medium. These results show that it is unlikely that the thermolytic action of neurotensin, at least within the hypothalamus, is mediated by the presynaptic release of dopamine.

Do central dopamine receptors have a physiological role in thermoregulation?

1 Core and tail skin temperature was measured in rats which had guide cannulae implanted into their brains to allow drug injections directly into the preoptic anterior hypothalamus. 2 Apomorphine and dopamine (10 microgram in 1 microliter) injected into the area of the preoptic anterior hypothalamus caused a fall in core temperature which was preceded by a rise in tail skin temperature. 3 The decrease in core temperature following central injection of either apomorphine or dopamine was significantly reduced by pretreating rats for 2 h with pikozide 0.5 mg/kg i.p.). 4 Bilateral intrahypothalamic injection of pimozide (0.5 microgram in 1 microliter) significantly reduced the hypothermic effect of systemic apomorphine (1.25 mg/kg i.p.). 5 Control rats placed 65 cm below a 250 W infrared lamp responded with vasodilation of tail skin blood vessels as indicated by an increase in tail skin temperature. Pimozide pretreatment (0.5 mg/kg i.p.) significantly reduced this response. 6 These results suggest that the preoptic anterior hypothalamus contains dopamine receptors which mediate hypothermia in rodents and raise the possibility that endogenous dopamine has a physiological role in thermoregulation.

Evidence for an endogenous dopamine-mediated hypothermia in the rat.

1 Unilateral intrahypothalamic injection of either dopamine (10 mug) or amphetamine (10 mug) caused a fall in core temperature in the rat. Pimozide (0.5 mg/kg, i.p.) significantly reduced the hypothermic response, whereas pretreatment with phentolamine (1 mg/kg, i.p.) or methysergide (5 mg/kg, i.p.) was ineffective.2 Systemic pretreatment with cocaine (20 mg/kg) abolished the hypothermic effect of amphetamine, but slightly enhanced the hypothermic response to dopamine.3 Systemic pretreatment with tranylcypromine (10 mg/kg) had no significant effect on the fall in core temperature induced by either amphetamine or dopamine.4 Intraperitoneal injection of cocaine and tranylcypromine, on their own, caused a fall in core temperature in the rat, which was significantly antagonized by either systemic or central pretreatment with pimozide. Phentolamine and methysergide failed to block the hypothermia.5 Unilateral intrahypothalamic injection of cocaine (20 mug) or tranylcypromine (10 mug) also caused a significant fall in core temperature, which was reduced by intrahypothalamic pretreatment with pimozide (0.5 mug), but not significantly changed by pretreatment with phentolamine (25 mug) or methysergide (5 mug).6 These results provide evidence for the presence of a dopaminergic system within the preoptic region, which mediates a lowering of core temperature in the rat.

Is acetylcholine involved in a dopamine receptor mediated hypothermia in mice and rats?

1 Apomorphine and oxotremorine caused a dose-related fall in core temperature in the mouse and a fall in core temperature preceded by an increase in tail-skin temperature in the rat.2 In both species the slope of the dose-response curve was greater for oxotremorine (8.2 +/- 0.6 mice, 0.9 +/- 0.1 rats) than it was for apomorphine (1.7 +/- 0.3 mice, 0.5 +/- 0.07 rats).3 The mouse was more sensitive than the rat to the effects of both agonists.4 Atropine (0.625 to 5 mg/kg) and hyoscine (0.5 and 1 mg/kg) caused a dose-related rightward shift of the dose-response curve to oxotremorine in mice, but pimozide (0.25 to 1 mg/kg) was ineffective. Similar results were obtained in the rat.5 Pimozide (0.125 to 1 mg/kg) caused a dose-related rightward shift of the dose-response curve to apomorphine in mice, but atropine (1.25 to 1 mg/kg) and hyoscine (0.5 and 1 mg/kg) were ineffective. Similar results were obtained in the rat.6 Intrahypothalamic injection of apomorphine (10 mug) and oxotremorine (1.25 mug) caused a fall in core temperature in rats. Pimozide (0.5 mg/kg i.p.) caused reversal of the effect of apomorphine but did not significantly change the response to oxotremorine. Atropine (2.5 mg/kg i.p.) blocked the effect of oxotremorine, but not that of apomorphine.7 These results suggest that there are both central dopamine and central muscarinic acetylcholine receptors which mediate a fall in core temperature in rodents, but do not support the hypothesis that any connection exists between these two receptor populations.

Different hypothalamic receptors mediate 5-hydroxytryptamine- and tryptamine-induced core temperature changes in the rat.

1 Unilateral intrahypothalamic injection of 5-hydroxytryptamine (5-HT) caused a dose-related fall in core temperature in rats, whereas injection of tryptamine into the same site caused a dose-related rise in core temperature. 2 The core temperature changes induced by 5-HT or tryptamine were inhibited by intrahypothalamic pretreatment with indoleamine receptor antagonists in a dose-related manner. 3 Other neurotransmitter antagonists, haloperidol, atropine, phentolamine and (-)-propranolol, had no significant effect on core temperature changes induced by 5-HT or tryptamine. 4 A differential antagonism was observed for the indoleamine receptor antagonists against 5-HT and tryptamine-induced core temperature changes. Methergoline and triflupromazine were more selective against tryptamine-induced hyperthermia, while cyproheptadine was more selective against 5-HT-induced hypothermia. 5 Intrahypothalamic pretreatment with 5,-7-dihydroxytryptamine (5,7-DHT) 42 nmol in 2 microliter inhibited tryptamine-induced hyperthermia, but was without effect on 5-HT-induced hypothermia. 6 These results suggest the possible existence of two different receptor populations within the preoptic anterior hypothalamus in rats; one specific for 5-HT and the other for tryptamine.

Involvement of a central dopaminergic system in 5-methoxy-N,N-dimethyltryptamine-induced turning behaviour in rats with lesions of the dorsal raphé nuclei.

The turning behaviour induced by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT) has been investigated in rats with lesions of the dorsal raphé nucleus (DRN). 5-MeODMT caused a dose-related contralateral turning in rats with 5,7-dihydroxytryptamine (5,7-DHT) lesions of the substantia nigra and a similar effect was observed in DRN-lesioned rats. In contrast, a dose-related ipsilateral turning was observed when 5-MeODMT was injected into rats with 5,7-DHT lesions of the striatum. These results suggest that the effects of 5-MeODMT in DRN-Lesioned rats are mediated via the substantia nigra. The contralateral turning induced by 5-MeODMT in rats with a 5,7-DHT lesion of the DRN was significantly reduced when a second 5-hydroxydopamine lesion was placed in the striatum, but not when it was placed in the nucleus accumbens. Thus the nigrostriatal dopaminergic system seems to be involved in 5-MeODMT-induced turning. The release of tritium from slices of substantia nigra previously labelled with [3H]-dopamine was inhibited by 5-MeODMT (10(-7) to 10(-5) M) and this effect was blocked by methysergide in a concentration-related manner. Tetrodotoxin (10(-7) M) failed to antagonise 5-MeODMT. These results suggest that 5-MeODMT can inhibit dopamine release from nigral dendrites, which could in turn enhance nigrostriatal activity by reducing the auto-inhibitory actions of dopamine, thereby causing contralateral turning in DRN-lesioned rats.

Complete genomic organization and promoter analysis of the round-spotted pufferfish JAK1, JAK2, JAK3, and TYK2 genes.

We have previously reported the isolation of the JAK1 gene from the round-spotted pufferfish. In the present study, we cloned and characterized genomic sequences encoding pufferfish JAK2, JAK3, and TYK2, which are other members of JAK family. To our knowledge, this is the first report to demonstrate the existence of four JAK genes in fish. All pufferfish JAK genes except JAK1 are composed of 24 exons; JAK1 has an additional exon. A comparison of the exon-intron organization of these genes revealed that the splice sites of JAK genes are nearly identical. In addition, all pufferfish JAK genes have one intron in the 5' untranslated region. Taken together, these data suggest that the pufferfish JAK genes may have evolved from a common ancestor. By 5' rapid amplification of cDNA ends and sequence analysis, we deduced the promoter regions for all JAK genes and found they do not contain typical TATA or CCAAT boxes but rather numerous other potential binding sites for transcription factors. Interestingly, the TYK2 gene is linked to CDC37 in a head-to-tail manner with a small intergenic region of 292 bp. Within this region, there are two potential binding sites for transcriptional factors such as c-Myb and NF-IL6. The putative promoter regions of all JAK genes were tested either in a carp CF cell line or in zebrafish embryos using CAT or lacZ as reporter genes. Both assays confirmed the transcriptional activities of these promoters in vitro and in vivo.

Enhancement of maximal thermogenesis by reducing endogenous adenosine activity in the rat.

Adenosine has been shown in vitro to be a potent antilipolytic agent and an inhibitor of insulin-stimulated glucose utilization in skeletal muscle. To test whether endogenously produced adenosine (e.g., from ATP hydrolysis) shares these deleterious effects on substrate mobilization and utilization and thus limits maximum thermogenesis in vivo, adenosine deaminase (converts adenosine to inosine) was given to rats 15 min before cold exposure. Significant (P less than 0.05) increases in thermogenesis were observed under both well-fed (100 units/kg ip) and food-rationed (200 units/kg ip) states. Significant (P less than 0.05) increases in thermogenesis and cold resistance were also observed after pretreatment with selective adenosine receptor antagonists [8-cyclopentyltheophylline (1 microgram/kg ip) greater than 1,3-dipropyl-8-p-sulfophenylxanthine (1.25 mg/kg ip) greater than aminophylline (18.7 mg/kg ip)], indicating an A1-receptor-mediated effect. These results indicate that endogenously released adenosine can indeed attenuate the thermogenic capacity in severe cold and that adenosine antagonists, especially those selective for A1-receptor, are useful in improving cold resistance under varying nutritional states.

In vivo release of dopamine during perfusion of neurotensin in substantia nigra of the unrestrained rat.

The functional effect of neurotensin on the kinetics of dopamine (DA) release in the substantia nigra of the freely moving rat was investigated. After guide tubes for push-pull perfusion were implanted stereotaxically just above the substantia nigra, endogenous stores of DA in this structure were labelled by micro-injection of 0.02-0.05 microCi of [14C]-DA. Then an artificial cerebrospinal fluid (CSF) was perfused within the site at a rate of 20 microliters/min at successive 5 min intervals. Neurotensin added to the CSF perfusate in concentrations of 0.05-0.1 microgram/microliter evoked an immediate, Ca++ dependent release of DA from sites directly within the substantia nigra or a delayed efflux when the peptide was perfused at the edge of this structure. Neurotensin failed to affect the pattern of release of this monoamine at sites which were not within the substantia nigra. Further, the body temperature of the rat also was not altered by neurotensin at any of the sites of perfusions. A relatively inactive analogue of the peptide, [D-Arg]9 neurotensin, was essentially without effect on DA activity. In double isotope experiments in which the substantia nigra of the rat was labelled with both [3H]-5-HT and [14C]-DA, the perfusion with neurotensin failed to affect 5-HT efflux while the release of DA was enhanced. Chromatographic analysis of the metabolites of DA in samples of push-pull perfusates revealed that neurotensin enhanced significantly the level of DOPAC and HVA. Overall, these results demonstrate that in the unrestrained rat neurotensin acts selectively within the substantia nigra to alter the presynaptic, Ca++ dependent release of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Is neurotensin in the brain involved in thermoregulation of the monkey?

Cannulae for intracerebroventricular (ICV) infusion were implanted stereotaxically in monkeys (Macaca fascicularis) maintained post-operatively in a primate restraint chair. During each experiment, a series of physiological measures was recorded simultaneously on a polygraph which included colonic temperature, vasomotor tone, heart rate, respiratory rate, and basal metabolism as reflected by O2 uptake. The ICV infusion in a volume of 0.5 ml of neurotensin (NT) in doses ranging from 3-150 micrograms produced neither a statistically significant nor consistent change in body temperature or vasomotor response. Although the highest dose of 450 micrograms NT infused ICV caused an immediate bradycardia and a concomitant decline in metabolic and respiratory rates, an average decline in core temperature of 0.6 degrees C and the accompanying cutaneous vasodilation often had a latency as long as 1.0 hr. In contrast to the typical hypothermia in this species following an ICV infusion of catecholamines, implicated in the central pathways underlying thermoregulation, NT failed to elicit a coordinated set of physiological responses for heat dissipation in the monkey. Therefore, it is unlikely that this tridecapeptide plays a role in the central mechanisms mediating the control of body temperature of this primate species.

In vivo microdialysis study on changes in septal dynorphin and beta-endorphin activities in active and hibernating Columbian ground squirrels.

State-dependent changes in extracellular concentration of endogenous opioids in the septum of Columbian ground squirrels were examined in the hibernating and euthermic states using in vivo microdialysis. The order of estimated extracellular concentration was found to be: hibernating > interbout euthermia > non-hibernating euthermia for dynorphin A and interbout euthermia > hibernating > non-hibernating euthermia for beta-endorphin. The apparent turnover rates of dynorphin A during hibernation was 15 times greater than that during euthermic non-hibernation phase and that of beta-endorphin was 8-fold greater. These results demonstrate that subfamilies of endogenous opioids may vary differentially in their activities at different stages of an annual hibernation cycle and may reflect their different roles in the regulation of hibernation.