Cerebrospinal fluid orexin-A levels in systemic lupus erythematosus patients presenting with excessive daytime sleepiness.

Objective Involvement of the hypothalamus is rare in patients with systemic lupus erythematosus (SLE). In this study, we measured cerebrospinal fluid (CSF) orexin-A levels in SLE patients with hypothalamic lesions to investigate whether the orexin system plays a role in SLE patients with hypothalamic lesions who present with excessive daytime sleepiness (EDS). Methods Orexin-A levels were measured in CSF from four patients with SLE who presented with hypothalamic lesions detected by MRI. Three patients underwent repeated CSF testing. All patients met the updated American College of Rheumatology revised criteria for SLE. Results Tests for serum anti-aquaporin-4 antibodies, CSF myelin basic protein and CSF oligoclonal bands were negative in all patients. All patients presented with EDS. Low to intermediate CSF orexin-A levels (92-180 pg/ml) were observed in three patients in the acute stage, two of whom (patients 1 and 2) underwent repeated testing and showed increased CSF orexin-A levels, reduced abnormal hypothalamic lesion intensities detected by MRI and EDS dissipation at follow-up. In contrast, CSF orexin-A levels were normal in one patient (patient 4) while in the acute stage and at follow-up, despite improvements in EDS and MRI findings. Patient 4 showed markedly increased CSF interleukin-6 levels (1130 pg/ml) and a slightly involved hypothalamus than the other patients. Conclusions Our findings suggest that the orexinergic system has a role in EDS in SLE patients with hypothalamic lesions. Furthermore, cytokine-mediated tissue damage might cause EDS without orexinergic involvement.

Environmental risk factors for REM sleep behavior disorder: a multicenter case-control study.

OBJECTIVE: Idiopathic REM sleep behavior disorder is a parasomnia characterized by dream enactment and is commonly a prediagnostic sign of parkinsonism and dementia. Since risk factors have not been defined, we initiated a multicenter case-control study to assess environmental and lifestyle risk factors for REM sleep behavior disorder. METHODS: Cases were patients with idiopathic REM sleep behavior disorder who were free of dementia and parkinsonism, recruited from 13 International REM Sleep Behavior Disorder Study Group centers. Controls were matched according to age and sex. Potential environmental and lifestyle risk factors were assessed via standardized questionnaire. Unconditional logistic regression adjusting for age, sex, and center was conducted to investigate the environmental factors. RESULTS: A total of 694 participants (347 patients, 347 controls) were recruited. Among cases, mean age was 67.7 ± 9.6 years and 81.0% were male. Cases were more likely to smoke (ever smokers = 64.0% vs 55.5%, adjusted odds ratio [OR] = 1.43, p = 0.028). Caffeine and alcohol use were not different between cases and controls. Cases were more likely to report previous head injury (19.3% vs 12.7%, OR = 1.59, p = 0.037). Cases had fewer years of formal schooling (11.1 ± 4.4 years vs 12.7 ± 4.3, p < 0.001), and were more likely to report having worked as farmers (19.7% vs 12.5% OR = 1.67, p = 0.022) with borderline increase in welding (17.8% vs 12.1%, OR = 1.53, p = 0.063). Previous occupational pesticide exposure was more prevalent in cases than controls (11.8% vs 6.1%, OR = 2.16, p = 0.008). CONCLUSIONS: Smoking, head injury, pesticide exposure, and farming are potential risk factors for idiopathic REM sleep behavior disorder.

Influence of aqueous extract of Agaricus blazei on rat liver toxicity induced by different doses of diethylnitrosamine.

The modifying potential of prior administration of an aqueous extract of the mushroom Agaricus blazei Murrill (Agaricaceae) (Ab) on hepatotoxicity induced by different doses of diethylnitrosamine (DEN) in male Wistar rats was evaluated. During 2 weeks, animals of groups G3 (Ab+DEN(50)), G5 (Ab+DEN(100)), G7 (Ab+DEN(200)), and G8 (Ab-treated) were treated with the A. blazei through drinking water. After this period, groups G2 (DEN(50)), G3 (Ab+DEN(50)), G4 (DEN(100)) G5 (Ab+DEN(100)), G6 (DEN(200)), and G7 (Ab+DEN(200)) were given a single i.p. injection of 50, 100 and 200 mg/kg of DEN, respectively, while groups G1 (non-treated) and G8 (Ab-treated) were treated with 0.9% NaCl only. All animals were killed 48 h after DEN or NaCl treatments. The hepatocyte replication rate was estimated by the index of the proliferating cell nuclear antigen (PCNA) positive hepatocytes and the appearance of putative preneoplastic hepatocytes through expression of the enzyme glutathione S-transferase placental form (GST-P). After DEN-treatment, ALT levels, PCNA labeling index, and the number of GST-P positive hepatocytes were lower in rats that received A. blazei treatment and were exposed to 100 mg/kg of DEN. Our findings suggest that previous treatment with A. blazei exerts a hepatoprotective effect on both liver toxicity and hepatocarcinogenesis process induced by a moderately toxic dose of DEN.

[A case of multiple sclerosis with hypothalamic amenorrhea].

We present a 31-year-old woman of multiple sclerosis. At age 28, she was admitted with complaints of echolalia and a gradual onset of weakness affecting the right upper and bilateral lower limbs. Brain MRI showed high intensity areas in the bilateral frontal gyri, lobuli paracentralis, and left anterior thalamus. Although she had been in remission for 3 years, she developed dysesthesia of left upper and lower limbs. Cervical T2 weighted MRI showed a new high signal intensity lesion in the spinal cord from the C2 to C3 level. The combination of the cerebral, thalamic and spinal cord lesions with remission and excerbations allowed the diagnosis of clinically MS to be made. She suffered amenorrhea from the onset of her illness. Serum prolactin was within the normal range. The LH and FSH basal secretions were decreased and there were low delayed secretions of LH and FSH after intravenous injection of 100 micrograms LHRH. We consider that her amenorrhea was caused by the hypothalamic lesion, supported by MR findings of dilatation of the third ventricle.

Effects of the mycelial extract of cultured Cordyceps sinensis on in vivo hepatic energy metabolism and blood flow in dietary hypoferric anaemic mice.

The beneficial effects of a traditional Chinese medicine, Cordyceps sinensis (Cs), on mice with hypoferric anaemia were evaluated by NMR spectroscopy. Experimental hypoferric anaemia was induced in mice by feeding with an Fe-free diet for 6 weeks. They were then given extract from cultured Cs (200 mg/kg body weight daily, orally) and were placed on an Fe-containing recovery diet (35 mg Fe/kg diet) for 4 weeks. In vivo 31P and 2H NMR spectra acquired noninvasively and quantitatively at weekly intervals were used to evaluate hepatic energy metabolism and blood flow in the mice. During the 4-week Cs-extract treatment, consistent increases were observed in liver beta-ATP: inorganic phosphate value by liver 31P NMR spectroscopy, representing the high energy state, and in blood-flow rate as determined by 2H NMR spectroscopy of deuterated water (D2O) uptake after intravenous injection of D2O. The haematological variables (the packed cell volume and the haemoglobin level) and the hepatic intracellular pH, which was determined from the NMR chemical shift difference between the inorganic phosphate peak and the alpha-phosphate peak of ATP, were not significantly different between Cs-extract-treated and control mice. As blood flow and energy metabolism are thought to be linked, the Cs-extract-increased hepatic energy metabolism in the dietary hypoferric anaemic mice was concluded to be due to increased hepatic blood flow.

Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus.

A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RIIalpha in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RIIalpha were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

Prenylflavonoids: a new class of non-steroidal phytoestrogen (Part 2). Estrogenic effects of 8-isopentenylnaringenin on bone metabolism.

In order to examine whether 8-isopentenylnaringenin (1), which has been proven to possess estrogen agonist activity in in vitro tests, also produces in vivo estrogenic properties, the effects of 1 on uterus and on bone metabolism were determined in ovariectomized rats. Rats were ovariectomized and treated with 1 at 30 mg/kg/day subcutaneously for two weeks or 17 beta-estradiol at 0.01 mg/kg/day subcutaneously for two weeks. Ovariectomy resulted in an increase in urinary excretion of bone resorption markers (hydroxyproline, pyridinoline and deoxypyridinoline) and a decrease in bone mineral density of the proximal tibia as well as reduced uterine weight. Treatment with 1 or 17 beta-estradiol completely suppressed these ovariectomy-induced bone and uterine changes in a qualitatively similar manner. These results demonstrate that 1 acts as an estrogen agonist in the uterus as well as in bone in vivo.

Contribution of sustained Ca2+ elevation for nitric oxide production in endothelial cells and subsequent modulation of Ca2+ transient in vascular smooth muscle cells in coculture.

To elucidate the intracellular Ca2+ (Ca2+i ) transient responsible for nitric oxide (NO) production in endothelial cells (ECs) and the subsequent Ca2+i reduction in vascular smooth muscle cells (VSMCs), we administrated four agonists with different Ca2+i-mobilizing mechanisms for both cells in iso- or coculture. We monitored the Ca2+i of both cells by two-dimensional fura-2 imaging, simultaneously measuring NO production as NO2-. The order of potency of the agonists in terms of the peak Ca2+i in ECs was bradykinin (100 nM) > ATP (10 microM) > ionomycin (50 nM) > thapsigargin (1 microM). In contrast, the order in reference to both the extent of Ca2+i reduction in cocultured VSMCs and the elevation in NO production over the level of basal release in ECs completely matched and was ranked as thapsigargin > ionomycin > ATP > bradykinin. Treatment by NG-monomethyl-L-arginine monoacetate but not indomethacin or glybenclamide restored the Ca2+i response in cocultured VSMCs to the isoculture level. In ECs, when the Ca2+ influx was blocked by Ni2+ or by chelating extracellular Ca2+, all four agonists markedly decreased NO production, the half decay time of the Ca2+i degenerating phase, and the area under the Ca2+i curve. The amount of produced NO hyperbolically correlated to the half decay time and the area under the Ca2+i curve but not to the Ca2+i peak level. Thus, the sustained elevation of Ca2+i in ECs, mainly a result of Ca2+ influx, determines the active NO production and subsequent Ca2+i reduction in adjacent VSMCs. Furthermore, L-arginine but not D-arginine or L-lysine at high dose (5 mM) without agonist enhanced the NO production, weakly reduced the Ca2+i in ECs, and markedly decreased the Ca2+i in VSMCs, demonstrating the autocrine and paracrine effects of NO (Shin, W. S., Sasaki, T., Kato, M., Hara, K., Seko, A., Yang, W. D., Shimamoto, N., Sugimoto, T., and Toyo-oka, T. (1992) J. Biol. Chem. 267, 20377-20382).

Inflammatory cytokine production induced by an analogue of muramyl dipeptide MDP-Lys(L18) in rat macrophage cultures and dog synovial fluid.

To examine the involvement of cytokines in the mechanisms of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine, MDP-Lys(L18)-induced arthritis, we analyzed interleukin-1 (IL-1), tumor necrosis factor (TNF), colony-stimulating factor (CSF), and neutrophil chemotactic factor (NCF) by bioassays in the rat macrophage-conditioned medium (Mluminal diameter-CM) stimulated by MDP-Lys(L18) in vitro and the synovial fluid from dogs treated subcutaneously with MDP-Lys(L18) for 14 days in vivo. The dog showed arthritis characterized by swelling of the knee joint, increased synovial fluid and thickened synovial membrane, and a single subcutaneous injection of MDP-Lys(L18) was previously shown to induced synovitis in rat tarsal joint. IL-1, TNF, CSF, and NCF activities in Mluminal diameter-CM were increased by MDP-Lys(L18), while only NCF activity was detected in the dog synovial fluid. Partial purification procedures revealed that NCF in Mluminal diameter-CM was not leukotriene B4 but a protein having heparin-affinity, and, in addition, immuno-reactive IL-8 was evident to be in Mluminal diameter-CM. The NCF activity in the dog synovial fluid was not inhibited by dialysis, showing that NCF is a protein substance, possibly a chemokine. These results suggest that MDP-Lys(L18) produces a chemokine, such as IL-8, which recruits neutrophils to the synovial membrane for subsequent development of synovitis in rats and dogs.

Pericholangitis with ulcerative colitis following autoimmune hepatitis over 12 years.

A 35-year-old woman was diagnosed as autoimmune hepatitis 12 years ago by abnormal findings of liver tests including lupus erythematosus (LE) cell phenomenon and liver biopsy. She was admitted in May 1990 with a history of lower gastrointestinal bleeding. Colonoscopy with biopsy and barium enema revealed chronic ulcerative colitis along the entire colon. Since liver tests did not respond well to prednisolone treatment, liver biopsy was again performed and it revealed periductal inflammation with small duct proliferation, a finding compatible with pericholangitis. We herein report this patient who was initially diagnosed as autoimmune hepatitis and thereafter found to be pericholangitis associated with ulcerative colitis.

Antitumor agents, 127. Bruceoside C, a new cytotoxic quassinoid glucoside, and related compounds from Brucea javanica.

Bruceoside C [5], a new quassinoid glucoside, and related compounds were isolated from Brucea javanica, and their structures were elucidated by spectral data. Bruceoside C showed potent cytotoxicities against KB, A-549, RPMI, and TE-671 tumor cells.

Survival-promoting effect of NGF on in vitro septohippocampal neurons with cholinergic and GABAergic phenotypes.

Recently, we demonstrated a survival-promoting effect of nerve growth factor (NGF) on cultured hippocampus-projecting neurons from developing septum/diagonal band region using fluorescent latex microspheres as a retrograde neuronal marker (Arimatsu et al., 1989). In the present study, we characterized these projection neurons by combining the retrograde cell labeling and histochemical stainings for acetylcholinesterase (AChE) activity and NGF receptor-, choline acetyltransferase- (ChAT-) and gamma-aminobutyric acid- (GABA-) immunoreactivities. The surviving microsphere-labeled neurons were, for the most part, immunoreactive for NGF receptor in the culture. A great majority (about 90%) of the microsphere-labeled neurons showed strong AChE activity and ChAT-immunoreactivity. The number of strongly AChE-positive neurons and that of ChAT-immunoreactive neurons in the culture supplemented with NGF was much greater with than without exogenous NGF. In addition, a major part (about 70%) of the microsphere-labeled neurons exhibited GABA-immunoreactivity in the presence of NGF. The number was also much greater than that without NGF. A considerable portion of cultured septal cholinergic neurons were shown to express GABA-immunoreactivity by a two-color immunofluorescence labeling experiment for ChAT and GABA. These findings are consistent with the assumption that NGF plays an important role in the development and organization of the cholinergic and GABAergic septohippocampal systems by supporting the neuronal survival, and raise a possibility that cholinergic and GABAergic fractions of the septohippocampal neurons may be developmentally correlated.

Effect of combined dopamine and bunazosin on the ischemic heart after occlusion of the coronary artery.

In order to investigate whether dopamine combined with bunazosin improves cardiac function, the global and regional cardiac function and regional blood flow of 7 anesthetized dogs were analyzed before and after occlusion of the left anterior descending coronary artery (LAD), then after 10 micrograms/kg/min dopamine infusion following the LAD occlusion, and again after a bolus infusion of bunazosin 250 micrograms/kg. Dopamine with bunazosin reduced left atrial pressure from 4.9 +/- 0.9 to 3.1 +/- 0.5 mmHg (p less than 0.05) and improved cardiac output from 1.22 +/- 0.15 to 1.50 +/- 0.14 L/min (p less than 0.05), maximum positive left ventricular dp/dt from 1721 +/- 202 to 3600 +/- 663 mmHg/sec (p less than 0.05) and the time constant from 45.2 +/- 5.0 to 27.5 +/- 4.6 msec (p less than 0.01). Bunazosin added to the dopamine reduced the elevated left ventricular peak systolic pressure caused by dopamine from 130 +/- 7 to 113 +/- 8 mmHg (p less than 0.01). With regard to the regional wall motion, the impaired LAD-delta L (the segment systolic shortening) and LAD-Elmax (the slope of peak systolic pressure--endsystolic length relation) following the LAD occlusion improved from 0.5 +/- 2.5 per cent to 5.9 +/- 2.6 per cent (p less than 0.01) and from 50 +/- 9 to 82 +/- 14 mmHg/mm (p less than 0.01) after the infusion of dopamine with bunazosin. Dopamine greatly increased the Rate Pressure Product (RPP) from 12610 +/- 1120 after LAD occlusion to 16950 +/- 1420, whereas dopamine in combination with bunazosin did not increase the RPP due to a drop of LV-PSP with little change in regional myocardial blood flow. It was concluded that combining dopamine with bunazosin was useful for improving both the global and regional cardiac functions of the ischemic heart.

Interleukin-6 as a neurotrophic factor for promoting the survival of cultured basal forebrain cholinergic neurons from postnatal rats.

Human recombinant interleukin-6 (IL-6, B-cell stimulating factor-2) was capable of supporting neuronal survival in cholinergic neuron culture, prepared from 10-day-old rat brain septal region. Cell survival of the cultured cholinergic neurons was estimated by measuring the remaining choline acetyltransferase (ChAT) activities after 6 days of culture. IL-6 at a concentration of 5 ng/ml maintained a more than 3-fold higher ChAT activity in the culture as compared with that in cultures without IL-6. The maximal dose of IL-6 was near 50 ng/ml. The concomitant addition of mouse nerve growth factor (NGF) and IL-6, both at maximal doses, had a synergistic effect on cholinergic cell survival. These results indicate that IL-6 can act as a neurotrophic agent, independent of the action of NGF, supporting neuronal survival of cultured postnatal rat septal cholinergic neurons. On the other hand, IL-6 did not affect the differentiation of the cultured embryonic rat septal cholinergic neurons, differently from the differentiation action by NGF.

Colocalization of NGF receptor immunoreactivity and [3H]GABA uptake activity in developing rat septum/diagonal band neurons in vitro.

Neurons in the septum (SEP) and vertical limb of the diagonal band (vDB) of 6-day-old rats were dissociated and cultured for 3 days. Cells with high affinity uptake mechanism for gamma-aminobutyric acid (GABA) were identified autoradiographically using 16 nM [3H]GABA. Cells that expressed nerve growth factor (NGF) receptor immunoreactivity were identified immunocytochemically using MC192, a monoclonal antibody against NGF receptor. Double labeling experiments combining [3H]GABA uptake with immunostaining for NGF receptor showed that 46% of the NGF receptor-immunoreactive cells accumulated [3H]GABA. The result was discussed in relation to a possible involvement of the developing septal GABAergic neurons in NGF-induced neuronal survival.

Effects of continuous infusion of cholinergic drugs on memory impairment in rats with basal forebrain lesions.

The effects of continuous infusion of cholinergic drugs on behavior in normal rats and on impaired acquisition and retention of several behavioral tasks in rats with basal forebrain (BF) lesions were investigated. Physostigmine and oxotremorine were infused continuously with a miniosmotic pump for 3 weeks, and the performance on several different behavioral tasks was examined during the infusion. In normal rats high doses of physostigmine (4 and 8 mg/kg/day s.c.) produced significant changes in general behavior and impaired performance in the Morris water maze. Oxotremorine (0.25-2 mg/kg/day s.c.) had no significant effects on general behavior or cognitive performance in normal rats, although severe cataracts developed at the high dose (4 mg/kg/day). A deficit in motor habituation in rats with BF lesions produced by bilateral injections of ibotenic acid (30 nmol on each side) was improved markedly by the chronic administration of physostigmine (2 mg/kg/day) and oxotremorine (1 mg/kg/day). BF lesions produced severe impairments in acquisition and retention in a passive avoidance task, an active avoidance and the Morris water maze, which was characterized by a marked disruption of retention. The impairment was also ameliorated markedly by the cholinergic drugs, whereas other behavioral impairments were not affected by the drugs. These results indicate that the continuous administration of cholinergic drugs produces a marked improvement of acquisition and retention in rats with BF lesions, and suggest that the impairment in cognitive performance, especially with regard to retention, caused by BF lesions is due to the disruption of the BF-cortical cholinergic pathway.

Ischemic myocardial protection. Comparison of nonoxygenated crystalloid, oxygenated crystalloid, and oxygenated fluorocarbon cardioplegic solutions.

This study was designed to compare myocardial protection with a nonoxygenated crystalloid solution, an oxygenated crystalloid solution, and an oxygenated fluorocarbon cardioplegic solution. Postischemic ventricular performance was studied in three equal (N = 7) groups of dogs subjected to 120 minutes of global ischemia induced at an average myocardial temperature of 18.5 degrees +/- 1.4 degrees C (range 17.0 degrees to 21.0 degrees C). Left ventricular global and regional function was evaluated by sonomicrometry and micromanometers before ischemia and at 45 and 60 minutes after ischemia. Stroke volume index, left ventricular pressure-minor external diameter loop area, percent shortening, first derivative of left ventricular pressure, mean velocity of circumferential fiber shortening, and the slope of the end-systolic pressure were used to evaluate myocardial contractility. In vitro oxygen content of the three cardioplegic solutions was measured at a mean injection temperature of 8.3 degrees +/- 0.6 degrees C: 0.8 +/- 0.1 vol% (nonoxygenated crystalloid cardioplegia), 3.2 +/- 0.2 vol% (oxygenated crystalloid cardioplegia), and 6.2 +/- 0.2 vol% (oxygenated fluorocarbon cardioplegia). Recovery of global and regional function was significantly (p less than 0.05) better with both oxygenated solutions than with the nonoxygenated solution. Differences between the oxygenated crystalloid and fluorocarbon groups were not significant. We conclude: (1) Compared to nonoxygenated crystalloid cardioplegia, oxygenated crystalloid and oxygenated fluorocarbon cardioplegic solutions gave superior myocardial protection during 2 hours of ischemic arrest; (2) no difference was found in protective effects between an oxygenated crystalloid and an oxygenated fluorocarbon solution.

A practical evaluation of accuracy with the standard deviation index (SDI) chart method. Second report. Report covering the period from April 1980 to March 1983.

To evaluate the accuracy of laboratory data for 18 items determined with an Technicon SMAC (Sequential Multiple Analyzer Computer), the SDI chart method was used in investigating the data according to five kinds of control survey programs from April 1980 to March 1983. The results can be summarized as follows: Inorganic phosphorus showed a gradual downward shift, arriving at the minus side of SDI in all control surveys. Potassium showed a particularly large fluctuation as was the case in the previous evaluation. The other items were relatively stable (i.e., showed less fluctuation), although many of them showed an upward shift. Some of the control survey programs yielded biased evaluation results. Thus, the SDI chart method proved to allow, as one of the external quality control techniques, observation of long-term, slight changes without necessitating any complicated calculation. Furthermore, it was confirmed that this is a practically useful method of accuracy evaluation because it allows overall evaluation of the data from various control survey programs on the same graph.