High-frequency signals (> 400 hz): a new window in electrophysiological analysis of the somatosensory system.

High-frequency signals (HFSs) between 400-1500 Hz in Magnetoencephalography (MEG) and Electroencephalography (EEG) provide a new window in electrophysiological analysis of the somatosensory system in humans and in other animals. The HFS in the primary somatosensory (SI) cortex precedes the conventional N20. In the swine model, they appear to be due to spiking in thalamocortical axonal terminals and in the soma and dendrites of cortical neurons. These spiking activities seem to activate slower conductances in the pyramidal cells in layers II-III and V, which give rise to N20. The HFS monitoring may be useful for separately evaluating the electrophysiology of the subcortical and cortical components of the somatosensory pathway.

Responsiveness of the cortical taste area neurons to a mixture of the four basic tastants in rats.

The taste coding mechanism in the cortical taste area was investigated by analyzing the responses of 59 neurons in the cortical taste area of the anesthetized rat to a mixture of the four basic tastants in both absence and presence of bicuculline methiodide, a specific antagonist to the GABA(A) receptors. The mixture caused response suppression more frequently than response facilitation, both in the control state and during bicuculline application. Cluster analysis revealed that only a group of the neurons with the best response to both NaCl and HCl (group NH) showed the best response to the mixture in the control state, whereas during bicuculline application, in addition to group NH, two other groups of neurons responding to sucrose, or to HCl and quinine responded vigorously to the mixture. Multidimensional scaling located the mixture outside the space of the four basic tastants facing an NaCl-HCl line in both states. GABAergic inhibition caused the group NH to represent the taste of the mixture in the control state. Thus, the mixture probably tastes salty and sour to rats. No cortical neuron was found which specifically responded to the mixture.

Benzylidene anabaseines act as high-affinity agonists for insect nicotinic acetylcholine receptors.

Benzylidene anabaseines are agonists selective for vertebrate alpha7-nicotinic acetylcholine receptor (nAChR), while they exhibit antagonist activity toward vertebrate alpha4beta2-nAChR. To investigate the effects of benzylidene anabaseines on insect nAChRs, we performed [3H]epibatidine-binding assays and neurophysiological experiments using American cockroach (Periplaneta americana) nerve-cord preparations. Of the six compounds tested, 3-benzylideneanabaseine (BA) and 6'-chloro-3-benzylideneanabaseine (CBA) displayed the highest potency in the binding assays, with K(i)s of 35.0 and 21.2nM, respectively. The introduction of a nitro group at the 4-position of the phenyl group led to a decrease in affinity by two orders of magnitude, while that of a chlorine atom at the 6'-position had little effect on affinity. In neurophysiological experiments, BA at 3.3 microg/ml increased the spike frequency observed with the nerve preparation, as observed with nicotine at 16.6 microg/ml. These findings suggest that benzylidene anabaseines act as high-affinity agonists in P. americana nAChRs and that they might therefore prove useful as probes for insect nAChRs.

Analysis of complex formation between crown ethers and potassium ion by determining retention factors in reversed-phase high-performance liquid chromatography.

Retention factors in reversed-phase high-performance liquid chromatography (RP-HPLC) were utilized for the analysis of complex formation reaction of four crown ethers with potassium ion in water-methanol media. The crown ethers were injected as analytes, potassium ion was present in the eluent, and three types of reversed-phase columns were used. The retention factor of the crown ethers decreased with increasing concentrations of potassium ion in the eluent, which is attributed to the change in the species of the crown ether from a neutral ligand to a positively charged complex. The complex formation constants were determined by analyzing the changes in the retention factor with a non-linear least-squares method.

Mechanisms of phytosterolemia in stroke-prone spontaneously hypertensive and WKY rats.

Analysis of sterol composition in serum, liver, adipose tissue, adrenals, and abdominal aorta demonstrated that the contents of plant sterols, campesterol and sitosterol, were evidently higher in WKY and stroke-prone spontaneously hypertensive (SHRSP) rats than in Wistar and WKA rats fed a diet containing a 0.5% plant sterol mixture. Lymphatic 24-hour recovery of 3H-sitosterol was about 2-fold higher in the WKY and SHRSP rats than in the WKA rats. Lymphatic absorption of 14C-cholesterol was also higher in WKY and SHRSP rats compared with WKA rats, but the difference was smaller than in the case of sitosterol. The remarkable increase of sitosterol absorption in WKY and SHRSP rats was observed between 9 and 24 hours after the administration. In SHRSP rats, lymphatic absorption of sitosterol between 0 and 3 hours was also higher than those in the other rat strains. Markedly less esterified 3H-sitosterol was detected in lymph than 14C-cholesterol in all strains, and in WKY and SHRSP rats, only a small increase in the esterified forms of sitosterol and cholesterol was observed. Although the incorporation of micellar 3H-sitosterol and 14C-cholesterol into intestinal brush border membranes was higher in SHRSP rats than in WKA rats, no difference was observed between WKY and WKA rats. These observations suggest that the incorporation into the brush border membranes and the esterification of sterols are not the major determinants for the hyperabsorption of sitosterol and cholesterol in SHRSP and WKY rats. Secretion of sitosterol and cholesterol in the bile of rats fed a plant sterol mixture was lower in SHRSP than in WKA rats. These results suggest that WKY and SHRSP strains deposit plant sterols in the body by enhancing the absorption and lowering the excretion of plant sterols. These strains of rats may be suitable models for studying mechanisms of differential absorption of various sterols.

Effect of dietary seal and fish oils on lipid metabolism in hamsters.

Eicosapentaenoic and docosahexaenoic acids were distributed mainly in the sn-1 and 3 positions of seal oil triacylglycerols and in the sn-2 position of fish oil triacylglycerols. Seal oil-rich or fish oil-rich fats having constant polyunsaturated (PUFAs)/monounsaturated/saturated fatty acids and n-6/n-3 PUFAs ratios were fed to hamsters for 3 weeks. The control fat contained linoleic acid as the sole PUFA. The concentration of triacylglycerols in the liver was significantly lower in the fish oil group than in the control group. Phospholipid concentration in serum was lower and that in the liver was higher in the seal oil group compared with the fish oil group. The activities of fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH), and the malic enzyme were significantly lower in both the fish and seal oil groups than in the control group. Dietary seal oil more effectively reduced arachidonic acid content in liver phosphatidylcholine and phosphatidylethanolamine and serum phosphatidylcholine than fish oil. These results showed that different intramolecular distribution of n-3 PUFAs influenced glycerolipid metabolism and arachidonic acid content in serum and liver phospholipids of hamsters.

Large-scale manufacturing of all four 2'-deoxynucleosides via novel strategies including a chemo-enzymatic process.

A chemical synthesis of 2-deoxyribose-1-phosphate 2 and its enzymatic conversion into purine 2'-deoxynucleosides (dNus) are shown. Besides the chemo-enzymatic process for purine dNus, a modified process for practical dC preparation is also established. Consequently, a series of practical manufacturing processes of all four dNus have been realized via novel strategies.

Effects of dietary defatted squid on cholesterol metabolism and hepatic lipogenesis in rats.

Male Sprague-Dawley rats were fed a cholesterol-free (Exp. 1) or cholesterol-supplemented (Exp. 2) diet containing 20% casein (control group) or 15% defatted squid and 5% casein (defatted squid group), as protein, for 14 d. Serum and hepatic cholesterol concentrations were lower in rats fed defatted squid than in those fed casein in both cholesterol-free (-20%, P < 0.05 and -15%, P < 0.05, respectively) and cholesterol-supplemented (-25%, P < 0.05 and -15%, P < 0.05, respectively) diets. Hepatic triglyceride concentration was lower in the defatted squid than in the control groups in both cholesterol-free (-51%, P< 0.05) and cholesterol-supplemented diets (-38%, P < 0.01). The activities of cytosolic fatty acid synthase and the NADPH-generating enzymes, malic enzyme and glucose-6-phosphate dehydrogenase, in the liver were lower in the defatted squid than in the control groups in both cholesterol-free (-21%, P< 0.01, -33%, P < 0.05, and -33%, P < 0.01, respectively) and cholesterol-supplemented diets (-34%, P < 0.05, -57%, P < 0.05, and -67%, P < 0.05, respectively). The activity of mitochondrial carnitine palmitoyltransferase in the liver was comparable between the control and defatted squid groups. The activity of Mg2+-dependent phosphatidate phosphohydrolase in the liver cytosol was lower in the defatted squid (-9%, P < 0.05) than in the control groups only in the cholesterol-free diet. Fecal excretion of total steroids was stimulated by the feeding of defatted squid in both cholesterol-free (+77%, P < 0.005) and cholesterol-supplemented diets (+29%, P < 0.01). These results suggest that the nonlipid fraction of squid exerts a hypocholesterolemic effect by increasing the excretion of total steroids in feces. The fraction also induces a triglyceride-lowering activity in the liver by decreasing hepatic lipogenesis.

[Study of neoadjuvant chemotherapy for invasive bladder cancer with MEC (methotrexate, epirubicin, cisplatin) therapy].

Eighteen patients with locally invasive bladder cancer were treated with 1 or 2 cycles of neoadjuvant chemotherapy consisting of methotrexate, epirubicin and cisplatin (MEC). All patients underwent radical cystectomy and pelvic lymph node dissection. Down-staging was observed in twelve (complete pathological response in 3 and partial pathological response in 9) patients (response rate were 67%). Four of the 18 patients died of disease and all of them had not achieved down-staging. Multivariate analysis revealed nodal status to be the only independent predictor. With regard to side effects, gastrointestinal symptoms and myelo-suppression were observed in almost all patients. Thrombocytopenia was observed in 13 patients (72%) including 7 patients who showed symptoms over grade 3. Gastro-intestinal symptoms and leukocytopenia disappeared with granisetron and granulocyte colony stimulating factor. There were no treatment-related deaths in this study. These results indicate that MEC therapy was safely performed and showed a high response rate in patients with locally invasive bladder cancer.

Reduced hepatic triglyceride secretion in rats fed docosahexaenoic acid-rich fish oil suppresses postprandial hypertriglyceridemia.

To evaluate the mechanisms of suppression of postprandial hypertriglyceridemia by fish oil rich in docosahexaenoic acid, the effect on the intestinal absorption of triglyceride, activities of lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) and metabolism of chylomicrons (CM) and CM remnants were compared with that of safflower oil in Sprague-Dawley rats in a series of studies. The feeding of fish oil for 3 wk suppressed postprandial hypertriglyceridemia (study 1). Dietary fish oil did not alter the rate of lymphatic absorption of triglyceride (study 2). The activities of LPL and HTGL were measured at 5 h after the beginning of feeding, when serum triglyceride concentrations were highest in both dietary groups. The activities of LPL in adipose tissue and heart were greater (P < 0.05) and those of HTGL were lower (P < 0.05) in the rats fed fish oil (study 3). In contrast, there were no differences in the activities of LPL and HTGL in postheparin plasma between the fish and safflower oil groups (study 4). The clearance rates of CM and CM remnants were measured by injecting intravenously CM collected from rats fed safflower or fish oils with [14C]triolein and [3H]cholesterol (study 5). Dietary oil did not influence the half-lives of CM or CM remnants. The secretion of triglyceride from the liver of rats injected with Triton WR-1339 was lower (P < 0.05) in the rats fed docosahexaenoic acid, a major component of fish oil, than those fed linoleic acid, a major component of safflower oil (study 6). These observations strongly support the hypothesis that in rats, the principal cause of the suppression of postprandial hypertriglyceridemia by fish oil is the depression of triglyceride secretion from the liver.

Development of chemo-enzymatic process and manufacture of deoxynucleosides.

Manufacturing method for 2'-deoxynucleosides (dNus) has been developed using novel chemo-enzymatic process. The method consists of the chemical synthesis of 2'-deoxyribose 1-phosphate (dRP) and the enzymatic conversions of dRP into 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG) and 2'-deoxycytidine (dC). Large-scale preparation is successfully done, which demonstrates an applicability of this strategy to practical use. As thymidine is already under commercial-scale production, all four dNus are now ready for scale-up.

Intestinal absorbability of wheat allergens, subunits of a wheat alpha-amylase inhibitor, expressed by bacteria.

Wheat CM2, CM3 and CM16 proteins are known as subunits of the tetrameric alpha-amylase inhibitor as well as major allergens to baker's asthma. The purpose of this study is to produce these CM proteins by bacteria in a quantity adequate for studying the penetration characteristics of the CM proteins through intestinal mucosa in rats and Caco-2 cells. cDNAs encoding the mature proteins were expressed in Escherichia coli and purified by an Ni2+-chelating column. The recombinant proteins were radioiodinated and admministered orally to rats or applied to the apical site of the Caco-2 cell monolayer. The radioactivity in the trichloroacetic acid-insoluble fraction, which was mainly composed of peptides with molecular mass less than that of the intact CM proteins, in the serum and the basolateral medium was highest in recombinant CM3. Accordingly, the intestinal absorption of these three proteins in the form present in wheat should be evaluated.

[A case of bladder tumor producing granulocyte-colony stimulation factor and parathyroid hormone-related protein].

A 68-year-old woman presented with urinary pain and frequency. Cystoscopy, intravenous pyelography and magnetic resonance imaging showed a huge bladder mass and hydronephrosis of the left kidney. Transurethral resection of bladder tumor (TUR-Bt) was done. Histopathological findings of TUR-biopsy was high grade transitional cell carcinoma. Post operatively, the laboratory examination showed marked leukocytosis with a maximum of 99,600/mm3 in the peripheral blood and a high level of granulocyte colony stimulating factor (G-CSF), 70 pg/ml in the serum (normal: less than 9.8 pg/ml). Serum calcium level increased gradually and parathyroid hormone-related protein (PTH-rP) revealed high, 8.4 pMol/l (normal: less than 0.6 pMol/l). The tumor cells were positive for G-CSF and PTH-rP immunohistochemical staining. She died of the disease 46 days after the operation. This is the third case of G-CSF and PTH-rP producing bladder tumor in the literature.

[5'-DFUR chemoprophylaxis in superficial bladder cancer. Kanagawa Urology 5'-DFUR Study Group].

5'-Deoxy-5-fluorouridine (5'-DFUR), an oral fluorinated pyrimidine carbamate, is widely used in patients with gastrointestinal and breast cancers because of its effectiveness. However, in bladder cancer, response rates have only been reported in Phase II clinical trials. Therefore, we conducted a prospective randomized trial to investigate chemoprophylactic effect of 5'-DFUR against recurrence of superficial bladder cancer after transurethral bladder tumor resection (TUR-Bt). The subjects were grouped as follows: 1) 5'-DFUR group (n = 31), received 600 mg/day of 5'-DFUR starting 2-3 weeks after TUR-Bt for 2 years; and 2) control group (n = 31) received no 5'-DFUR. Although there was no significant difference between groups, the cumulative recurrence rates was more favorable in the 5'-DFUR group (p = 0.256) than in the controls. Results according to cancer factors showed that, in patients with G2 based on grading, those in the 5'-DFUR group tended to have a lower recurrence rate than the control group (p = 0.070). There was a 40% incidence of adverse drug reactions (12/30 patients), primarily slight gastrointestinal symptoms which disappeared or improved with drug discontinuation. The results of the present study suggest that 5'-DFUR might be the choice of treatment to prevent recurrence of superficial bladder cancer.

Significance of soluble TNF receptor-I in acute-type fulminant hepatitis.

OBJECTIVE: Fulminant hepatitis is associated with apoptosis of hepatocytes, which is mediated via Fas and tumor necrosis factor (TNF) receptors. The clinical significance of apoptotic factors and these receptors was investigated in fulminant hepatitis. METHODS: Serum levels of TNF-alpha, soluble TNF receptor-I and -II, soluble Fas antigen, and Fas ligand were measured. Then, the relationships between these parameters and the severity or prognosis of fulminant hepatitis were studied. RESULTS: Serum levels of TNF-alpha, soluble TNF receptor-I, and soluble TNF receptor-II were increased in acute-type fulminant hepatitis. In particular, soluble TNF receptor-I was significantly higher than in patients with subacute-type fulminant hepatitis, severe acute hepatitis, acute hepatitis, or healthy controls. The soluble TNF receptor-I level continued to increase or remained high in patients who died of acute-type fulminant hepatitis, and eight of nine patients had a level >10 ng/ml. In contrast, the soluble TNF receptor-I level remained <10 ng/ml in survivors. Soluble Fas and soluble Fas ligand levels tended to increase in all types of acute liver disease and were not specific to fulminant hepatitis. CONCLUSION: Our findings suggested that monitoring the soluble TNF receptor-I level may help to assess the prognosis of acute-type fulminant hepatitis and that TNF might be associated with massive hepatic necrosis.

Significance of Fas antigen-mediated apoptosis in human fulminant hepatic failure.

OBJECTIVE: The aim of this study was to elucidate the role of apoptosis in human fulminant hepatic failure. We studied the expression of Fas antigen on liver tissues, Fas ligand in lymphocytes, and soluble Fas ligand in patients' serum. METHODS: On finding apoptotic cells in fulminant hepatic failure liver, we first examined them using the TUNEL method. Subsequently, the expression of Fas was studied by immunostaining. Simultaneously, Fas ligand presenting on both liver-infiltrated cells and peripheral lymphocytes was studied by reverse transcription-polymerase chain reaction, and soluble Fas ligand in sera was measured by ELISA. RESULTS: By using the TUNEL method, we first demonstrated that many apoptotic cells existed in fulminant hepatic failure but not in normal ones. Our immunohistochemistry study showed that many hepatocytes in fulminant hepatic failure strongly expressed Fas. In addition, Fas ligand on both liver-infiltrating lymphocytes and peripheral lymphocytes in fulminant hepatic failure patients was detected. The serum level of soluble Fas ligand was significantly increased in fulminant hepatic failure (mean value, 2.91 ng/ml in fulminant hepatic failure [n = 10], 1.62 ng/ml in acute hepatitis [n = 10], and 0.27 ng/ml in healthy controls [n = 10]). Furthermore, this serum level of sFas ligand was significantly associated with prothrombin time both in acute hepatitis and fulminant hepatic failure. CONCLUSIONS: The present results indicate that Fas-mediated apoptosis may be one of the triggers for the induction of fulminant hepatic failure.

Protection against the diabetogenic effect of feeding tert-butylhydroquinone to rats prior to the administration of streptozotocin.

We determined whether an oral administration of the synthetic antioxidant, tert-butylhydroquinone (TBHQ), or the naturally occurring lipoxygenase inhibitor, curcumin, to rats would provide protection against the diabetogenic effect of streptozotocin (STZ). Male Sprague-Dawley rats were fed on an AIN-76-based purified diet containing 0.0028% TBHQ or on the purified diet with a daily intragastric administration of curcumin (200 mg/kg of body weight) for one week while receiving intravenously administered STZ. The rats fed on the TBHQ-containing diet were resistant to diabetes development when compared with the rats fed on the TBHQ-free diet and had a higher body weight gain and lower serum glucose concentration. Glucose-stimulated insulin secretion from the pancreatic islet in the rats that had received TBHQ was higher than that in the control rats. The rats receiving curcumin showed no beneficial effect on these diabetic symptoms. These findings provide direct evidence for the suggestion that dietary supplementation of an antioxidant may exert a preventive effect on the diabetogenic action of free-radical producers.

Application of the random arbitrary primed polymerase chain reaction differential display method to isolate genes of cholesterol metabolism-related proteins from rat liver.

The random arbitrary primed (RAP) polymerase chain reaction (PCR) differential display (DD) method was applied to isolate genes related to cholesterol metabolism from exogenously hypercholesterolemic (ExHC) rats and the progenitor, SD rats. Forty-seven trials of RAP-PCR DD resulted in the isolation of 37 clones differing in strain, cholesterol supplementation or their interaction. Among their fingerprints, five clones gave reproducible patterns by a Northern blotting analysis. The sequence of two clones with lower mRNA abundance in ExHC rats than in SD rats was homologous to that of fatty acid synthase and oxalyl-CoA decarboxylase. Two other clones with higher mRNA on the n-cholesterol diet were matrin F/G protein and the NMDA receptor glutamate-binding subunit. The other clone with higher mRNA abundance in ExHC rats on the cholesterol diet was myelodysplasia/myeloid leukemia factor 2. Fifteen trials of reverse transcriptase (RT)-PCR DD yielded 10 clones, but none of the fingerprints were reproduced by the Northern blotting analysis. These results indicate that RAP-PCR DD is an appropriate alternative to RT-PCR DD for isolating the genes involved in hypercholesterolemia.