All-trans retinoic acid displays multiple effects on the growth, lipogenesis and adipokine gene expression of AML-I preadipocyte cell line.

Adipose tissue is a potential site of retinoic acid (RA) action, but its physiological significance remains to be clarified. We have examined the effect of all-trans retinoic acid (ATRA) on growth and differentiation of preadipocytes, and on adipokine gene expression in mature adipocytes using human preadipocyte cell model, AML-I. Both ATRA and 9-cis RA induced growth arrest in AML-I preadipocyte at between 50 and 100 µM, which was accompanied by apoptosis. Western blotting showed a loss of NF-κB, Bcl-2 and p-Akt, and the accumulation of Bad and Akt in cytoplasm of ATRA-treated AML-I preadipocytes. Exposure of AML-I to ATRA or 9-cis RA increased intracellular lipid accumulation in a time-dependent manner compared to vehicle-treated cells. Expression of fatty acid synthase (FAS) and peroxisome proliferator-activated receptor-γ (PPAR-γ) proteins was increased in ATRA-treated cells. Thus, both ATRA and 9-cis RA promoted differentiation, inhibited proliferation and induced apoptosis in AML-I preadipocytes. ATRA also modulated adipokine expression by increasing the mRNA level of adipocytokines (adiponectin, leptin and LPL), and by inhibiting PAI-1 mRNA expression in mature AML-I adipocytes. The data suggest that ATRA exerts a wide range of effects--growth arrest, apoptosis, lipogenesis and modulation of adipokine gene expression--during the maturation of preadipocytes into adipocytes.

Central nervous system lymphoma newly developed 12 years after remission of an ocular adnexal lymphoma.

Recurrence of non-Hodgkin's lymphoma more than 5 years after the initial diagnosis is rare. When late relapse occurs, it is difficult to determine whether it is a true recurrence or a new lesion. We experienced a case of an 81-year-old woman who developed central nervous system (CNS) lymphoma 12 years after remission of ocular adnexal lymphoma. Both showed the histology of diffuse large B-cell lymphoma. To elucidate whether the CNS lymphoma was clonally related to the first lymphoma, rearrangement of the immunoglobulin heavy chain genes of each lymphoma was studied using a polymerase chain reaction-based method. The results revealed that the sizes of the amplified products of the rearranged regions from the two lymphomas were different. This suggested different clonal origins of the lymphomas. It is clinically important to determine the origin of a second neoplasm because patients with a clonally related second lymphoma are usually treated with more intensive regimens, while those with a clonally unrelated lymphoma receive standard first-line therapy. The present case shows that, in the case of recurrent non-Hodgkin's lymphoma, not only histological confirmation but also genetic assessment is important to clarify the origin of the second lymphoma.

[Effective treatment with tranexamic acid for chronic disseminated intravascular coagulation associated with aortic dissection].

An 82-year-old female with a history of aortic dissection was admitted to our hospital for evaluation of thrombocytopenia and a bleeding tendency. Blood coagulation test data demonstrated that the patient had disseminated intravascular coagulation (DIC) secondary to aortic dissection. We initiated anti-fibrinolytic therapy with tranexamic acid. After this anti-fibrinolytic therapy, thrombocytopenia and the levels of fibrinogen and fibrinogen degradation products improved. In addition, we reviewed 7 other patients who were treated with tranexamic acid for DIC associated with aortic dissection. Anti-fibrinolytic therapy with tranexamic acid is effective for treating DIC secondary to aortic dissection.

[Acute upper airway obstruction after extubation due to excessive anteflexion of the neck with occipitocervical fusion].

A patient developed upper airway obstruction immediately after tracheal extubation due to excessive anteflexion of the neck with occipitocervical fusion. A 59-year-old woman who had undergone mastectomy 17 years previously was scheduled for occipitocervical fusion for C2 vertebral metastasis. Retroflexion of her neck was restricted. Nasal intubation under sedation was performed using bronchial fiberscopy under fentanyl and propofol anesthesia. Emergence from anesthesia was smooth, and extubation was performed. Immediately after extubation, the patient could not breathe, and manual mask ventilation was impossible. Re-intubation was performed 30 minutes after the extubation. Oral fiberscopy revealed pharyngeal obstruction, and laryngeal edema was not observed. Fixation of her neck in excessive anteflexion was suspected to have caused her dyspnea. Therefore, re-operation was performed, and she was transferred to the intensive care unit under anesthesia. One day postoperatively, extubation was performed successfully with no dyspnea. Fixation of the neck in excessive anteflexion is one of the causes of upper airway obstruction after occipitocervical fusion. We must carefully observe cervical X-ray films to locate the upper airway obstruction, and careful extubation using a tube exchanger is strongly recommended in this operation.

[A case of femoral neuropathy after radical ovariectomy].

We experienced a 55-year-old female patient who was diagnosed as femoral neuropathy after radical ovariectomy. An epidural catheter was introduced at T11-12 interspace without any problems and general anesthesia was induced and maintained. The operation ended uneventfully. On the first postoperative day, she noticed hypesthesia of the inner surface of her left thigh and could not raise the left leg. The symptom remained after the removal of epidural catheter on the second postoperative day, and the influence of insertion of the epidural catheter on the symptom was suspected. We performed neurological examinations and found weakness of the left quadriceps femoris muscle, weakness of the left patellar reflex, and weakness of touch sensation and cold sensation and hypalgesia on the anterior surface of the left thigh and the inner surface of the left lower leg. Those findings led us to diagnose with femoral neuropathy probably due to abdominal retractors or the operation itself, and insertion of epidural anesthesia could not be the cause of neuropathy. Her symptom was ameliorated with a conservative therapy after four months. We should perform fine neurological examinations when neurological complications occur, especially when we use epidural catheters, and also should have the knowledge about those complications.

Induction of apoptosis and lipogenesis in human preadipocyte cell line by n-3 PUFAs.

We examined the effect of n-3 PUFAs (polyunsaturated fatty acids) on the growth and maturation of human preadipocyte cell line AML-I. On day 3 of the culture, n-3 fatty acids such as DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), but not n-6 fatty acid LA (linoleic acid), induced growth arrest accompanied by the appearance of characteristics of apoptosis in AML-I cells at concentrations between 250 and 500 µM by Annexin V-FITC staining. In Western blotting analysis, the loss of NF-κB, Bcl-2 and p-Akt and the accumulation of Bad and Akt were observed in the cytoplasmic protein from the EPA-treated cells. Exposure of AML-I to EPA or DHA increased the cytoplasmic lipid accumulation compared with the vehicle-treated cells in a time-dependent manner during 4 and 6 days culture period by Oil Red O staining. The expression of FAS (fatty acid synthase) and PPAR-γ (peroxisome proliferator-activated receptor-γ) were increased in EPA-treated cells. These results suggest that EPA and DHA promote differentiation, inhibit proliferation and induce apoptosis in preadipocyte cell line AML-I.

The mechanisms of the direct action of etomidate on vascular reactivity in rat mesenteric resistance arteries.

BACKGROUND: Etomidate minimally influences hemodynamics at a standard induction dose in young healthy patients, but can cause significant systemic hypotension at higher doses for induction or electroencephalographic burst suppression (i.e., cerebral protection) in patients with advanced age or heart disease, and during cardiopulmonary bypass. However, less is known about its action on systemic resistance arteries. METHODS: Using an isometric force recording method and fura-2-fluorometry, we investigated the action of etomidate on vascular reactivity in small mesenteric arteries from young (7-8 wk old, n = 179) and aged (96-98 wk old, n = 10) rats. RESULTS: In the endothelium-intact strips from young rats, etomidate enhanced the contractile response to norepinephrine or KCl (40 mM) at 3 microM but inhibited it at higher concentrations (>or=10 microM). The enhancement was still observed after treatment with N(G)-nitro l-arginine, tetraethylammonium, diclofenac, nordihydroguaiaretic acid, losartan, ketanserin, BQ-123, or BQ-788, but was not observed in aged rats. In the endothelium-denuded strips from young rats, etomidate (>or=10 microM) consistently inhibited the contractile response to norepinephrine or KCl without enhancement at 3 microM. In the fura-2-loaded, endothelium-denuded strips from young rats, etomidate inhibited norepinephrine- or KCl-induced increases in both intracellular Ca(2+) concentration ([Ca(2+)]i) and force. Etomidate still inhibited the norepinephrine-induced increase in [Ca(2+)]i after depletion of the intracellular Ca(2+) stores by ryanodine, which was sensitive to nifedipine. Etomidate had little effect on norepinephrine- or caffeine-induced Ca(2+) release from the intracellular stores or Ca(2+) uptake into the intracellular stores. During stimulation with norepinephrine or KCl, etomidate had little effect on the [Ca(2+)]i-force relation at low concentrations (

Anesthetic management of a patient with aortocaval fistula.

Aortocaval fistula is a rare complication of ruptured abdominal aortic aneurysm (AAA), and patients with an aortocaval fistula show multiple symptoms. We report an 87-year-old man who was diagnosed as having an AAA with aortocaval fistula and who developed refractory hypotension after induction of anesthesia. Following a phenylephrine injection for slight hypotension induced by anesthetic induction, he developed severe hypotension and bradycardia, and his skin became cyanotic. Vasopressor agents had no immediate effect on the hypotension, but blood pressure gradually increased in about 30 min with continuous infusion of dopamine and noradrenaline. Transesophageal echocardiography (TEE) showed right ventricle (RV) hypokinesis and massive tricuspid regurgitation (TR). Central venous pressure (CVP) showed a remarkably high value. After the repair of the aortocaval fistula, the hemodynamics became stable, RV motion was improved, TR was reduced, and CVP became normal. Anesthetic management of the repair of an aortocaval fistula is very difficult. The hemodynamics changed dramatically throughout anesthesia in our patient with this disorder, even though low-dose anesthetics were used. For the successful treatment of this disorder, preparation for the operation is required before the induction of anesthesia, and urgent closure of the fistula is necessary after the induction of anesthesia. TEE is a useful tool for monitoring hemodynamics in such patients.

Sustained elevation of serum cortisol level causes sensitization of coronary vasoconstricting responses in pigs in vivo: a possible link between stress and coronary vasospasm.

Vasospastic angina is induced by stress, for which cortisol secreted by activated hypothalamic/pituitary/adrenal axis may play an important role. However, direct evidence for this notion is still lacking. In this study, we examined whether sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses in pigs in vivo and, if so, whether Rho-kinase, which we found is a key molecule of coronary vasospasm, is involved. Oral administration of cortisol (20 mg/kg per day) increased its serum level to that seen in restraint stress in pigs. Thus, we examined coronary vasomotor responses in the following 4 groups: (1) control (without cortisol); (2) cortisol (20 mg/kg per day, p.o.) for 9 days; (3) cortisol plus RU38486 (a glucocorticoids receptor antagonist, 10 mg/kg per day, p.o.) for 9 days; and (4) cortisol for 9 days followed by 6-week withdrawal. Coronary angiography showed that intracoronary serotonin caused coronary hyperconstriction and reduction in coronary blood flow associated with ischemic ECG changes (coronary vasospasm) in only the cortisol group. All of these responses were abolished by hydroxyfasudil, a specific Rho-kinase inhibitor, in vivo. Organ chamber experiments demonstrated that serotonin concentration-dependently caused hypercontractions of coronary vascular smooth muscle associated with Rho-kinase activation (as evidenced by the enhanced phosphorylation of myosin binding subunit, a substrate of Rho-kinase) in only the cortisol group. All of these responses were again inhibited by hydroxyfasudil in vitro. These results indicate that sustained elevation of serum cortisol level sensitizes coronary vasoconstricting responses through Rho-kinase activation, suggesting the link between stress and coronary vasospasm.

Ferulic acid attenuated cognitive deficits and increase in carbonyl proteins induced by buthionine-sulfoximine in mice.

beta-Amyloid peptide (Abeta), the major constituent of the senile plaques observed in the brains of Alzheimer's disease patients, is cytotoxic to neurons and plays a central role in the pathogenesis of this disease. Previous studies have suggested that oxidative stress is involved in the mechanisms of Abeta-induced neurotoxicity in vivo. Here, we used a mouse model of brain dysfunction induced by dl-buthionine-(S,R)-sulfoximine (BSO: 3micromol/3microL/mouse, i.c.v.), an inhibitor of glutathione synthesis. In the novel object recognition test, we found impairments of exploratory preference in the retention trial but not the training trial 24h after BSO treatment, suggesting that BSO produces cognitive dysfunction in mice. In the forebrain of this model, we observed increase in carbonyl protein levels, an index of biochemical oxidative damage of proteins, compared to vehicle-treated mice. Pretreatment with ferulic acid (5mg/kg, s.c.) once a day for 6 days inhibited the induction of deficits in memory and increase in carbonyl protein levels by BSO. These findings suggest that pretreatment with FA may attenuate the memory deficits and increase the carbonyl protein levels induced by BSO in mice.

Roles of endothelial oxidases in endothelium-derived hyperpolarizing factor responses in mice.

The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDHF in animals and humans and that superoxide anions derived from endothelial nitric oxide synthases (NOSs) system are an important precursor for EDHF/H2O2 in mice. There are several intracellular sources of superoxide anions other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, and mitochondrial electron transport chain. In this study, we examined the possible role of endothelial oxidases other than NOSs in the EDHF-mediated responses. In angiotensin II-infused mice, both EDHF-mediated relaxations and hyperpolarizations to acetylcholine were significantly reduced, nitric oxide-mediated relaxations were rather enhanced, and vascular smooth muscle responses were preserved. Antihypertensive treatment normalized blood pressure but failed to improve EDHF-mediated responses in those mice. Acute inhibition of endothelial oxidases other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, or mitochondrial electron transport chain, had no inhibitory effects on EDHF-mediated responses. Furthermore, in p47phox-knockout mice, EDHF-mediated responses were unaltered. These results suggest that endothelial oxidases other than NOSs are not involved in EDHF/H2O2 responses in mice, suggesting a specific link between endothelial NOSs system and EDHF responses under physiological conditions.

Pivotal role of Cu,Zn-superoxide dismutase in endothelium-dependent hyperpolarization.

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several vasodilating factors, including prostacyclin, NO, and endothelium-derived hyperpolarizing factor (EDHF). We have recently identified that endothelium-derived H2O2 is an EDHF in mesenteric arteries of mice and humans and in porcine coronary microvessels. However, the mechanism for the endothelial production of H2O2 as an EDHF remains to be elucidated. In this study, we tested our hypothesis that Cu,Zn-superoxide dismutase (Cu,Zn-SOD) plays a pivotal role in endothelium-dependent hyperpolarization, using control and Cu,Zn-SOD-/- mice. In mesenteric arteries, EDHF-mediated relaxations and hyperpolarizations were significantly reduced in Cu,Zn-SOD-/- mice with no inhibitory effect of catalase, while endothelium-independent relaxations and hyperpolarizations were preserved. Endothelial H2O2 production also was significantly reduced in Cu,Zn-SOD-/- mice. In Langendorff isolated heart, bradykinin-induced increase in coronary flow was significantly reduced in Cu,Zn-SOD-/- mice, again with no inhibitory effect of catalase. The exogenous SOD mimetic tempol significantly improved EDHF-mediated relaxations and hyperpolarizations and coronary flow response in Cu,Zn-SOD-/- mice. These results prove the novel concept that endothelial Cu,Zn-SOD plays an important role as an "EDHF synthase" in mice, in addition to its classical role to scavenge superoxide anions.

Growth arrest and apoptosis induced by quercetin is not linked to adipogenic conversion of human preadipocytes.

Quercetin is involved in several biological activities including inhibition of cell growth and induction of apoptosis in cancer cells. However, it is unclear and unknown whether quercetin influences cell maturation. We examined the effect of quercetin on the growth and differentiation of human preadipocyte cells AML-I. Induced growth arrest of AML-I by quercetin was accompanied by the appearance of characteristics of apoptosis under the adipogenic stimulation by annexin V-fluorescein isothiocyanate staining method. A decrease of nuclear factor-kappaB and the antiapoptotic protein Mcl-1 and an increase of the proapoptotic protein Bad were observed in time-dependent fashion in the quercetin-treated cells compared with the vehicle-treated cells by Western blot analysis. Structure-related flavonoids, including rutin (quercetin-3-O-rutinoside) and quercitrin (quercetin-3-O-rhamnoside), did not have any cytotoxic effect on AML-I. Interestingly, exposure of AML-I to quercetin for 6 days increased the amount of cytoplasmic lipid droplets as well as the expression of fatty acid synthase and peroxisome proliferator-activated receptor gamma proteins. These results suggested that apoptosis induced by quercetin was not linked to adipogenic conversion of preadipocytes.

Effects of pre-germinated brown rice on depression-like behavior in mice.

We investigated the antidepressant-like effects of pre-germinated brown rice (PGBR) and polished rice (PR) pellets, respectively, in comparison with control (AIN-93G) pellets in the forced swimming test and the learned helplessness paradigm in mice. Mice were fed respective pellets for 30 days. The immobility time on the 2nd day of the forced swimming test was shorter in mice fed with PR or PGBR pellets than in mice fed with control pellets. In the learned helplessness paradigm, the number of escape failures in mice fed with PGBR pellets was significantly smaller than that in mice fed with control pellets. Compared to the control group, an increase in serotonin (5-HT) levels, but not in 5-hydroxyindoleacetic acid (5-HIAA) levels, and a decrease in the 5-HIAA/5-HT ratio were observed in the frontal cortex of the PGBR group. There were no differences among the three groups in terms of 5-HT and 5-HIAA levels and their ratios in the hippocampus and striatum. The levels of noradrenaline and 3-methoxy-4-hydroxyphenylglycol were not affected by the food pellets in all the brain regions tested. Additionally, we could not detect any differences in the expression of the 5-HT1A receptor and the 5-HT transporter in the frontal cortex of the three groups. These results suggest that the increase of 5-HT levels in the mouse frontal cortex contributes to the antidepressant-like effects of PGBR pellets.

Long-term treatment with a Rho-kinase inhibitor improves monocrotaline-induced fatal pulmonary hypertension in rats.

Primary pulmonary hypertension is a fatal disease characterized by endothelial dysfunction, hypercontraction and proliferation of vascular smooth muscle cells (VSMCs), and migration of inflammatory cells, for which no satisfactory treatment has yet been developed. We have recently demonstrated that intracellular signaling pathway mediated by Rho-kinase, an effector of the small GTPase Rho, is involved in the pathogenesis of arteriosclerosis. In the present study, we examined whether the Rho-kinase-mediated pathway is also involved in the pathogenesis of fatal pulmonary hypertension in rats. Animals received a subcutaneous injection of monocrotaline, which resulted in the development of severe pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions in 3 weeks associated with subsequent high mortality rate. The long-term blockade of Rho-kinase with fasudil, which is metabolized to a specific Rho-kinase inhibitor hydroxyfasudil after oral administration, markedly improved survival when started concomitantly with monocrotaline and even when started after development of pulmonary hypertension. The fasudil treatment improved pulmonary hypertension, right ventricular hypertrophy, and pulmonary vascular lesions with suppression of VSMC proliferation and macrophage infiltration, enhanced VSMC apoptosis, and amelioration of endothelial dysfunction and VSMC hypercontraction. These results indicate that Rho-kinase-mediated pathway is substantially involved in the pathogenesis of pulmonary hypertension, suggesting that the molecule could be a novel therapeutic target for the fatal disorder.

Long-term inhibition of Rho-kinase suppresses angiotensin II-induced cardiovascular hypertrophy in rats in vivo: effect on endothelial NAD(P)H oxidase system.

Intracellular signaling pathway mediated by small GTPase Rho and its effector Rho-kinase plays an important role in regulation of vascular smooth muscle contraction and other cellular functions. We have recently demonstrated that Rho-kinase is substantially involved in angiotensin II-induced gene expressions and various cellular responses in vitro. However, it remains to be examined whether Rho-kinase is involved in the angiotensin II-induced cardiovascular hypertrophy in vivo and, if so, what mechanisms are involved. Long-term infusion of angiotensin II for 4 weeks caused hypertrophic changes of vascular smooth muscle and cardiomyocytes in rats. Both changes were significantly suppressed by concomitant oral treatment with fasudil, which is metabolized to a specific Rho-kinase inhibitor, hydroxyfasudil, after oral administration. Angiotensin II caused a perivascular accumulation of macrophages and Rho-kinase activation, both of which were also significantly suppressed by fasudil. Vascular NAD(P)H oxidase expression (nox1, nox4, gp91phox, and p22phox) and endothelial production of superoxide anions were markedly increased by angiotensin II, both of which were also significantly suppressed by fasudil. Thus, fasudil ameliorated the impaired endothelium-dependent relaxations caused by angiotensin II without affecting vasodilator function of vascular smooth muscle. These results provide evidence that Rho-kinase is substantially involved in the angiotensin II-induced cardiovascular hypertrophy in rats in vivo. The suppression of endothelial NAD(P)H oxidase upregulation and resultant superoxide production and the amelioration of endothelial vasodilator function may be involved in this process.

Endothelium-derived hydrogen peroxide accounts for the enhancing effect of an angiotensin-converting enzyme inhibitor on endothelium-derived hyperpolarizing factor-mediated responses in mice.

UNLABELLED: Background- We have recently identified that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans, for which endothelial nitric oxide synthase (eNOS) is an important source. Angiotensin-converting enzyme (ACE) inhibitors are known to enhance EDHF-mediated responses. In this study, we examined whether endothelium-derived H2O2 accounts for the enhancing effect of an ACE inhibitor on EDHF-mediated responses and, if so, what mechanism is involved. METHODS AND RESULTS: Control and eNOS-/- mice were maintained with or without temocapril (10 mg/kg per day orally) for 4 weeks, and isometric tensions and membrane potentials of mesenteric arteries were recorded. In control mice, temocapril treatment significantly enhanced EDHF-mediated relaxations and hyperpolarizations to acetylcholine (n=8 each). Catalase, a specific scavenger of H2O2, abolished the beneficial effects of temocapril, although it did not affect endothelium-independent relaxations to sodium nitroprusside or NS1619, a direct opener of K(Ca) channels (n=6 each). Western blot analysis demonstrated that the temocapril treatment significantly upregulated the expression of eNOS. By contrast, this enhancing effect of temocapril was absent in eNOS-/- mice (n=6). CONCLUSIONS: These results indicate that endothelium-derived H2O2 accounts for the enhancing effect of temocapril on EDHF-mediated responses caused in part by eNOS upregulation, further supporting our H2O2 theory.

Synergistic effect of fosfomycin and arbekacin on a methicillin-resistant Staphylococcus aureus-induced biofilm in a rat model.

Biofilms are a major concern for clinicians in the treatment of infectious disease because of the resistance to a wide range of antibiotics. Using a rat air pouch model, methicillin-resistant Staphylococcus aureus (MRSA) growing as a biofilm was treated with a combination of fosfomycin (FOM) and arbekacin (ABK) or by the agents alone. This model has the advantage of permitting frequent sampling of exudates for bacterial counts and anti-bacterial activity, and morphological examination of the biofilm structure and inflammatory process in the pouch tissues. A clear synergistic effect was observed in the rats treated with a combination of fosfomycin and arbekacin. Morphological studies using scanning electron microscopy and histological staining showed dramatic changes of the biofilm structure as well as the inflammatory response in the rats. These results suggested an enhancement of bactericidal activity of arbekacin penetrating through the biofilm layer by virtue of fosfomycin. A possible mechanism of the synergistic effect is discussed.