Systemic administration of α-lipoic acid suppresses excitability of nociceptive wide-dynamic range neurons in rat spinal trigeminal nucleus caudalis.

Although a modulatory role has been reported for α-lipoic acid (LA) on T-type Ca2+ channels in the nervous system, the acute effects of LA in vivo, particularly on nociceptive transmission in the trigeminal system, remain to be determined. The aim of the present study was to investigate whether acute intravenous LA administration to rats attenuates the excitability of wide dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. Extracellular single unit recordings were made from seventeen SpVc neurons in response to orofacial mechanical stimulation of pentobarbital-anesthetized rats. Responses to both non-noxious and noxious mechanical stimuli were analyzed in the present study. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was significantly and dose-dependently inhibited by LA (1-100 mM, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 5 min. These inhibitory effects lasted for approximately 10 min. These results suggest that acute intravenous LA administration suppresses trigeminal sensory transmission, including nociception, via possibly blocking T-type Ca2+ channels. LA may be used as a therapeutic agent for the treatment of trigeminal nociceptive pain.

Serum Polyunsaturated Fatty Acid Composition and Serum High-Sensitivity C-Reactive Protein Levels in Healthy Japanese Residents: The KOBE Study.

BACKGROUND/OBJECTIVES: Serum polyunsaturated fatty acid (PUFA) composition reflects dietary intake and is related to risks for cardiovascular diseases. We hypothesized that serum n-3 PUFA composition, especially including long-chain n-3 PUFA such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), is associated with inflammatory status, which is related to increased risk for cardiovascular diseases. SUBJECTS/METHODS: We investigated the relationship between serum PUFA composition and high-sensitivity C-reactive protein (hs-CRP) levels in a cross-sectional study among 1,102 healthy men and women aged 40-74 years who reside in Kobe City. Multiple linear regression models that predict hs-CRP level were prepared to confirm the contribution of serum total n-3 PUFA, long-chain n-3 PUFA, EPA and DHA compositions after adjusting for other PUFAs and atherosclerotic risk factors. RESULTS: The serum n-3 PUFA, particularly long-chain n-3 PUFA, compositions were inversely associated with the hs-CRP levels. The standardized regression coefficient was -0.089 (p < 0.01) for total n-3 PUFA, -0.091 (p < 0.01) for long-chain n-3 PUFA, -0.071 (p = 0.03) for EPA, and -0.068 (p = 0.04) for DHA. The n-6 PUFA compositions were also inversely associated with the hs-CRP levels (-0.169 [p < 0.01] for total n-6 PUFA and -0.159 [p < 0.01] for linoleic acid). CONCLUSIONS: The serum n-3 PUFA compositions were inversely related with the hs-CRP levels, similar associations were also observed in n-6 PUFA compositions. Our results suggest that dietary PUFA intake was inversely associated with attenuated inflammation in healthy Japanese population.

Anomalous magnetization behaviour in a single crystal of vanadium spinel FeV2O4.

Spinel oxide FeV2O4, having the orbital degrees of freedom at Fe(2+) and V(3+) ions, exhibits multi-step magnetic phase transitions and successive structural phase transitions at low temperatures. In order to clarify the magnetic properties of FeV2O4, we have measured the temperature dependence of magnetization, isothermal magnetization curves and specific heat using a single crystal of FeV2O4. Temperature-induced magnetization jumps below the 110 K were observed in the zero-field-cooled magnetization curves. Furthermore, we found that the behaviours of the isothermal magnetization curves were quite different between the zero-field-cooled and field-cooled conditions. We suggest that the change of the magnetic domain structure under the magnetic field associated with the orbital states of Fe(2+) ions is the possible origin of these intriguing and anomalous magnetic properties in a single crystal of FeV2O4.

Chemical synthesis and biological activity of rat INSL3.

The recently identified protein, insulin 3 (INSL3), has structural features that make it a bona fide member of the insulin superfamily. Its predicted amino acid sequence contains the classic two-peptide chain (A- and B-) structure with conserved cysteine residues that results in a disulphide bond disposition identical to that of insulin. Recently, the generation of insl3 knockout mice has demonstrated that testicular descent is blocked due to the failure of a specific ligament, the gubernaculum, to develop. The mechanism by which INSL3 exerts its action on the gubernaculum is currently unknown. The purpose of this study was to, for the first time, synthesize rat INSL3 and test its action on organ cultures of foetal rat gubernaculum. INSL3 also contains a cassette of residues Arg-X-X-X-Arg within the B-chain, a motif that is essential for characteristic activity of another related member of the superfamily, relaxin. Hence, the relaxin activity of rat INSL3 was also tested in two different relaxin bioassays. The primary structure of rat INSL3 was determined by deduction from its cDNA sequence and successfully prepared by solid phase peptide synthesis of the two constituent chains followed by their combination in solution. Following confirmation of its chemical integrity by a variety of analytical techniques, circular dichroism spectroscopy confirmed the presence of high beta-turn and alpha-helical content, with a remarkable spectral similarity to the synthetic ovine INSL3 peptide and to synthetic rat relaxin. The synthetic rat INSL3 bound with very low affinity to rat relaxin receptors and had no activity in a relaxin bioassay. Furthermore, it did not augment or antagonize relaxin activity. The rat INSL3 did however induce growth of foetal rat gubernaculum in whole organ cultures demonstrating that INSL3 has a direct action on this structure.

Ginkgo biloba extract-induced relaxation of rat aorta is associated with increase in endothelial intracellular calcium level.

Ginkgo biloba extract (GBE) has been used clinically for improving peripheral vascular diseases in France and Germany. In the present study, to clarify the pharmacological properties of vasodilation produced by GBE, we examined the effect of GBE and quercetin, one of the ingredients in GBE, on the thoracic aorta isolated from Wistar rats. GBE produced a dose-dependent relaxation in the aortic ring precontracted with noradrenaline, and the relaxation was abolished by L-N(G)-nitro arginine methyl ester (L-NAME). Quercetin produced a similar relaxation, which was also abolished by L-NAME. We then examined the effects of GBE and quercetin on the intracellular calcium level ([Ca2+]i) of cultured aortic endothelial cells using a fluorescent confocal microscopic imaging system. Both GBE and quercetin produced significant increases in [Ca2+]i in the endothelial cells. The increase in [Ca2+]i by quercetin (10(-6) M) was abolished by removing the extracellular Ca2+, but was not affected by thapsigargin, a calcium pump inhibitor. These findings suggest that a principal ingredient of GBE producing vasodilation is quercetin, which can activate nitric oxide synthesis and release by increasing [Ca2+]i in vascular endothelial cells.

Frontal midline theta rhythm is correlated with cardiac autonomic activities during the performance of an attention demanding meditation procedure.

Frontal midline theta rhythm (Fm theta), recognized as distinct theta activity on EEG in the frontal midline area, reflects mental concentration as well as meditative state or relief from anxiety. Attentional network in anterior frontal lobes including anterior cingulate cortex is suspected to be the generator of this activity, and the regulative function of the frontal neural network over autonomic nervous system (ANS) during cognitive process is suggested. However no studies have examined peripheral autonomic activities during Fm theta induction, and interaction of central and peripheral mechanism associated with Fm theta remains unclear. In the present study, a standard procedure of Zen meditation requiring sustained attention and breath control was employed as the task to provoke Fm theta, and simultaneous EEG and ECG recordings were performed. For the subjects in which Fm theta activities were provoked (six men, six women, 48% of the total subjects), peripheral autonomic activities were evaluated during the appearance of Fm theta as well as during control periods. Successive inter-beat intervals were measured from the ECG, and a recently developed method of analysis by Toichi et al. (J. Auton. Nerv. Syst. 62 (1997) 79-84) based on heart rate variability was used to assess cardiac sympathetic and parasympathetic functions separately. Both sympathetic and parasympathetic indices were increased during the appearance of Fm theta compared with control periods. Theta band activities in the frontal area were correlated negatively with sympathetic activation. The results suggest a close relationship between cardiac autonomic function and activity of medial frontal neural circuitry.

Determination of the spatial distribution of major elements in the rat brain with X-ray fluorescence analysis.

An energy dispersive X-ray fluorescence analysis was applied for determining the spatial (two-dimensional) distribution of elemental concentrations in rat brain sections. Freeze-dried brain sections prepared from normal and ischemic rats with middle cerebral artery occlusion were scanned with a collimated X-ray beam (0.18 mm in diameter, 50-kV acceleration voltage). The fluorescent Kalpha X-rays of P, S, Cl, and K were detectable, so that the two-dimensional distribution of fluorescent X-ray intensities could be determined for these elements. Furthermore, quantitative determination was possible for P and K by using the fundamental parameter technique. However, the accurate determination of Na and Ca was difficult, because of the low energy of Kalpha X-ray of Na, and the interference of K-Kbeta with Ca-Kalpha. The change in elemental concentrations in ischemic tissue, including the decrease in K concentration and increase in Cl concentration, was demonstrated by this method as a two-dimensional contour map. Since it is possible to obtain a pictorial representation of the elemental concentration in tissue sections, this method may be useful to evaluate the ionic changes in injured brain tissue in relation to histological or autoradiographical observations.

Serum-free coculture of stromal and epithelial cells from benign prostatic hyperplasia with keratinocyte growth factor.

We developed a serum-free coculture model of benign prostatic hyperplasia (BPH) to clarify whether stromal cells stimulate growth of epithelial cells from BPH tissues. Epithelial and stromal cells from freshly isolated BPH tissue were cultured separately in defined serum-free WAJC 404/RPMI 1640 medium supplemented with insulin, transferrin, selenium, hydrocortisone, bovine serum albumin, epidermal growth factor, basic fibroblast growth factor and keratinocyte growth factor. (3)H-Tdr incorporation into epithelial cells and stromal cells was used as a measure of proliferation. When epithelial cells were cocultured with stromal cells, (3)H-Tdr incorporation into epithelial cells was increased in comparison to that in epithelial cells cultured alone. Dihydrotestosterone significantly increased this effect. It is likely that the in vitro coculture model reported here will be useful for isolating and understanding stromal cell-derived paracrine growth factor(s).

Disturbance of consciousness associated with hypophosphatemia in a chronically alcoholic patient.

A 69-year-old man with chronic alcoholism was admitted to our hospital due to disturbance of consciousness and oliguria. Emergency laboratory examination revealed metabolic acidosis, hypoglycemia, hyponatremia, mild liver dysfunction, acute renal failure and rhabdomyolysis. After administration of fluids and nutrients and continuous hemodiafiltration, he recovered from all signs and symptoms except for disturbance of consciousness after 7 days. Since severe hypophosphatemia persisted, we administered adequate phosphates, and then his level of consciousness normalized. We discuss the relationships among alcohol abuse, hypophosphatemia and disturbance of consciousness, and recommend that hypophosphatemia be considered a potential cause of disturbance of consciousness in alcoholic patients.

The inhibitory effect of glabridin from licorice extracts on melanogenesis and inflammation.

Glabridin is the main ingredient in hydrophobic fraction of licorice extract affecting on skins. In this study, we investigated inhibitory effects of glabridin on melanogenesis and inflammation using cultured B16 murine melanoma cells and guinea pig skins. The results indicated that glabridin inhibits tyrosinase activity of these cells at concentrations of 0.1 to 1.0 microg/ml and had no detectable effect on their DNA synthesis. Combined analysis of SDS-polyacrylamide gel electrophoresis and DOPA staining on the large granule fraction of these cells disclosed that glabridin decreased specifically the activities of T1 and T3 tyrosinase isozymes. It was also shown that UVB-induced pigmentation and erythema in the skins of guinea pigs were inhibited by topical applications of 0.5% glabridin. Anti-inflammatory effects of glabridin in vitro were also shown by its inhibition of superoxide anion productions and cyclooxygenase activities. These data indicated that glabridin is a unique compound possessing more than one function; not only the inhibition of melanogenesis but also the inhibition of inflammation in the skins. By replacing each of hydroxyl groups of glabridin with others, it was revealed that the inhibitory effect of 2'-O-ethyl glabridin was significantly stronger than that of 4'-O-ethyl-glabridin on melanin synthesis in cultured B16 cells at the concentration of 1.0 mg/ml. With replacement of both of two hydroxyl groups, the inhibitory effect was totally lost. Based on these data, we concluded that two hydroxyl groups of glabridin are important for the inhibition of melanin synthesis and that the hydroxyl group at the 4' position of this compound is more closely related to melanin synthesis.

Absence of rectoanal inhibitory reflex in a child with multiple endocrine neoplasia type 2B.

BACKGROUND: Patients with multiple endocrine neoplasia (MEN) type 2B inherently present with gastrointestinal motility disorders as well as medullary thyroid carcinoma, adrenal pheochromocytoma, and Marfanoid habitus. METHODS: The authors examined gut motility function in a 7-year-old girl with MEN type 2B who had suffered from chronic constipation and recurrent acalculous cholecystitis since infancy. RESULTS: Results of total gastrointestinal barium meal and enema studies showed marked hypoperistalsis of the gut and entire colonic dilatation. Histopathologic study results of the gut wall from the stomach, duodenum, and rectum showed hyperplasia of the submucosal and intramuscular neural plexuses in all specimens. Anorectal manometry demonstrated disarrangement of the internal sphincter rhythmic wave and a complete absence of the rectoanal inhibitory reflex. CONCLUSION: These data suggest that gut motility disorders in MEN type 2B are caused by inadequately organized autonomic nervous system activity that originates from hyperplasia of the enteric nervous system.

Neurochemical features and synaptic connections of large physiologically-identified GABAergic cells in the rat frontal cortex.

Physiological and morphological properties of large non-pyramidal cells immunoreactive for cholecystokinin, parvalbumin or somatostatin were investigated in vitro in the frontal cortex of 18-22-day-old rats. These three peptides were expressed in separate populations including large cells. Cholecystokinin cells and parvalbumin cells made boutons apposed to other cell bodies, but differed in their firing patterns in response to depolarizing current pulses. Parvalbumin cells belonged to fast-spiking cells. Parvalbumin fast-spiking cells also included chandelier cells. In contrast, cholecystokinin cells were found to be regular-spiking non-pyramidal cells or burst-spiking non-pyramidal cells with bursting activity from hyperpolarized potentials (two or more spikes on slow depolarizing humps). Large somatostatin cells belonged to the regular-spiking non-pyramidal category and featured wide or ascending axonal arbors (wide arbor cells and Martinotti cells) which did not seem to be apposed to the somata so frequently as large cholecystokinin and parvalbumin cells. For electron microscopic observations, another population of eight immunohistochemically-uncharacterized non-pyramidal cells were selected: (i) five fast spiking cells including one chandelier cell which are supposed to contain parvalbumin, and (ii) three large regular-spiking non-pyramidal cells with terminals apposed to somata, which are not considered to include somatostatin cells, but some of which may belong to cholecystokinin cells. The fast-spiking cells other than a chandelier cell and the large regular-spiking non-pyramidal cells made GABA-positive synapses on somata (4% and 12% of the synapses in two small to medium fast-spiking cells, 22% and 35% of the synapses in two large fast-spiking cells, and 10%, 18% and 37% of the synapses in three large regular-spiking non-pyramidal cells). A few terminals of the fast-spiking and regular-spiking non-pyramidal cells innervated GABAergic cells. About 30% of the fast-spiking cell terminals innervated spines, but few of the regular-spiking non-pyramidal cell terminals did. A fast-spiking chandelier cell made GABA-positive synapses on GABA-negative axon initial segments. These results suggest that large GABAergic cells are heterogeneous in neuroactive substances, firing patterns and synaptic connections, and that cortical cells receive heterogeneous GABAergic somatic inputs.

GABAergic cell subtypes and their synaptic connections in rat frontal cortex.

Physiological, morphological and immunohistochemical characteristics of non-pyramidal cells in frontal cortex of young rats were studied in vitro by whole-cell recording and biocytin injection. Several groups of GABAergic non-pyramidal cells were identified: (i) parvalbumin fast-spiking (FS) cells with low input resistances and spikes of short duration, including extended plexus (basket) cells and chandelier cells. These cells showed abrupt episodes of non-adapting repetitive discharges; (ii) late-spiking (LS) cells exhibiting slowly developing ramp depolarizations, including neurogliaform cells; (iii) the remaining groups contained both burst-spiking (BS) or regular-spiking (RS) non-pyramidal (NP) cells. BSNP cells exhibited bursting activity (two or more spikes on slow depolarizing humps) from hyperpolarized potentials. Both these physiological types corresponded to a range of morphologies: (i) somatostatin-containing Martinotti cells with ascending axonal arbors to layer I (some were also positive for calbindin D28k); (ii) VIP-containing double bouquet cells with descending axonal arbors as well as arcade cells (these included small cells immunoreactive for CCK or calretinin). Each subtype of cells made GABAergic synapses onto relatively specific portions of cortical cells, but similar domains were innervated by multiple classes of GABA cells.

In vitro and in vivo antichlamydial activities of HSR-903, a new fluoroquinolone antibiotic.

The in vitro and in vivo antichlamydial activities of HSR-903 were investigated. The MICs of HSR-903 for different species of chlamydia were 0.016 to 0.063 microg/ml, which were superior to those of conventional fluoroquinolones. The therapeutic effect of HSR-903 in experimental mouse Chlamydia psittaci pneumonia was also excellent and almost equal to that of minocycline and superior to that of ofloxacin. These results indicate that HSR-903 may be useful in the treatment of respiratory infections caused by chlamydiae.

Two distinct subgroups of cholecystokinin-immunoreactive cortical interneurons.

Examination of cholecystokinin-immunoreactive cells in the rat frontal cortex revealed the presence in layers I-VI of a non-uniform population ranging in size from small to large. All were also immunoreactive for GABA. The most commonly observed dendritic form of the small cells were bipolar or bitufted although some were multipolar and demonstrated vasoactive intestinal polypeptide and in a few case calretinin immunoreactivity. The large cells were multipolar or bitufted and lacked expression of vasoactive intestinal polypeptide and calretinin immunoreactivity but occasionally showed calbindin D28k immunoreactivity. Therefore, the cholecystokinin-immunoreactive cells could be divided into two distinct subpopulations depending on their chemistry and morphology. Our previous studies showed that GABAergic cells in the neocortex could be classified into at least three chemically different subgroups: (1) parvalbumin-containing cells; (2) somatostatin-containing cells (most of them also contain calbindin D28k); and (3) vasoactive intestinal polypeptide- and/or calretinin-containing cells. The present results indicated that the small cholecystokinin-immunoreactive non-pyramidal cells constitute a subset of the vasoactive intestinal polypeptide- and/or calretinin-containing cortical GABAergic cells. The large cells remain to be categorized.

Pure silicate fragment in a recurrent stone former of calcium oxalate.

Long-time oral intake of magnesium-silicate-containing drugs is thought to be a causative factor inducing silicate urolithiasis. Besides, magnesium seems to play an anti-urolithogenic part in the formation of calcium oxalate stones. We report a recurrent calcium oxalate former who had been treated with magnesium aluminometasilicate antacid for gastric ulcer for approximately 17 years. One of the fragments found during extracorporeal shock wave lithotripsy was identified as 100% silicate. Deposition of silica was also found on other fragments. A large dose of magnesium (given in a part of the drug) might have little influence on the formation of calcium oxalate.

Enzymatic synthesis, isolation, and analysis of novel alpha- and beta-galactosyl-cycloisomalto-octaoses.

Novel branched cycloisomalto-octaoses (CI8s) were enzymatically synthesized by transgalactosylation with alpha-galactosidase from coffee bean and beta-galactosidase preparations from Penicillium multicolor and Bacillus circulans, using melibiose and lactose as donor substrates, and CI8 which is a cyclic homogeneous oligosaccharide composed of eight glucose units bound by alpha-(1-->6)-linkages, as an acceptor. alpha-Galactosyl-CI8s and beta-galactosyl-CI8s obtained were isolated and purified by HPLC. Their structures were elucidated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOFMS) and NMR spectroscopy.