Parvalbumin-, substance P- and calcitonin gene-related peptide-immunopositive axons in the human dental pulp differ in their distribution of varicosities.

Information on the frequency and spatial distribution of axonal varicosities associated with release of neurotransmitters in the dental pulp is important to help elucidate the peripheral mechanisms of dental pain, mediated by myelinated versus unmyelinated fibers. For this, we investigated the distribution of axonal varicosities in the human dental pulp using light- and electron-microscopic immunohistochemistry for the vesicular glutamate transporter 2 (VGLUT2), which is involved in the glutamatergic transmission, and syntaxin-1 and synaptosomal nerve-associated protein 25 (SNAP-25), combined with parvalbumin (PV), which is expressed mostly in myelinated axons, and substance P (SP) and calcitonin gene-related peptide (CGRP), which are expressed mostly in unmyelinated axons. We found that the varicosities of the SP- and CGRP-immunopositive (+) axons were uniformly distributed throughout the dental pulp, whereas those of PV+ axons were only dense in the peripheral pulp, and that the expression of PV, VGLUT2, syntaxin-1, SNAP-25, SP and CGRP was significantly higher in the varicosities than in the axonal segments between them. These findings are consistent with the release of glutamate and neuropeptides by axonal varicosities of SP+ and CGRP+ unmyelinated fibers, involved in pulpal pain throughout the human dental pulp, and by varicosities of PV+ fibers, arising from parent myelinated fibers, and involved in dentin sensitivity primarily in the peripheral pulp.

Distribution of excitatory and inhibitory axon terminals on the rat hypoglossal motoneurons.

Detailed information about the excitatory and inhibitory synapses on the hypoglossal motoneurons may help understand the neural mechanism for control of the hypoglossal motoneuron excitability and hence the precise and coordinated movements of the tongue during chewing, swallowing and licking. For this, we investigated the distribution of GABA-, glycine (Gly)- and glutamate (Glut)-immunopositive (+) axon terminals on the genioglossal (GG) motoneurons by retrograde tracing, electron microscopic immunohistochemistry, and quantitative analysis. Small GG motoneurons (< 400 µm2 in cross-sectional area) had fewer primary dendrites, significantly higher nuclear/cytoplasmic ratio, and smaller membrane area covered by synaptic boutons than large GG motoneurons (> 400 µm2). The fraction of inhibitory boutons (GABA + only, Gly + only, and mixed GABA +/Gly + boutons) of all boutons was significantly higher for small GG motoneurons than for large ones, whereas the fraction of Glut + boutons was significantly higher for large GG motoneurons than for small ones. Almost all boutons (> 95%) on both small and large GG motoneurons were GABA + , Gly + or Glut + . The frequency of mixed GABA +/Gly + boutons was the highest among inhibitory boutons types for both small and large GG motoneurons. These findings may elucidate the anatomical substrate for precise regulation of the motoneuron firing required for the fine movements of the tongue, and also suggest that the excitability of small and large GG motoneurons may be regulated differently.

Ultrastructural analysis of low-threshold mechanoreceptive vibrissa afferent boutons in the cat trigeminal caudal nucleus.

Ultrastructural parameters related to synaptic release and their correlation with synaptic connectivity were analyzed in the low-threshold mechanoreceptive vibrissa afferent boutons in laminae III and IV of the trigeminal caudal nucleus (Vc). Rapidly adapting vibrissa afferents were intra-axonally labeled, and quantitative ultrastructural analyses with serial sections were performed on the labeled boutons and their presynaptic endings (p-endings). The volume of the labeled boutons was widely distributed from small to large ones (0.8~12.3 µm(3)), whereas the p-endings were small and uniform in size. The volume of the labeled boutons was positively correlated with the ultrastructural parameters such as mitochondrial volume (correlation coefficient, r=0.96), active zone area (r=0.82) and apposed surface area (r=0.79). Vesicle density (r=-0.18) showed little correlation to the volume of labeled boutons, suggesting that the total vesicle number of a bouton is proportional to its volume. In addition, the bouton volume was positively correlated with the number of p-endings (r=0.52) and with the number of dendrites postsynaptic to the labeled bouton (r=0.83). These findings suggest that low-threshold mechanoreception conveyed through vibrissa afferents is processed in a bouton size-dependent manner in the Vc, which may contribute to the sensory-motor function of laminae III/IV in Vc.

Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study.

A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.

Saucernetin-7 isolated from Saururus chinensis induces caspase-dependent apoptosis in human promyelocytic leukemia HL-60 cells.

In the present study, we investigated the effect of saucernetin-7 (a biologically active compound isolated from the underground parts of Saururus chinensi) on the induction of apoptosis and the putative pathways of its action in HL-60 human promyelocytic leukemia cells. Saucernetin-7-treated HL-60 cells displayed several features of apoptosis, including DNA fragmentation, DNA laddering by agarose gel electrophoresis, and externalization of annexin-V targeted phosphatidylserine (PS) residues. z-VAD-fmk (a broad-caspase inhibitor) almost completely suppressed saucernetin-7-induced DNA ladder formation, thereby implicating the caspase cascade in the apoptotic process. We also observed that saucernetin-7 caused the activations of caspase-3, -8 and -9, and that it induced Bid cleavage, the mitochondrial translocation of Bax from the cytosol, and cytochrome c release from mitochondria, but it had no effect on Bcl-2 and Bcl-xL levels. Taken together, the present study demonstrates that saucernetin-7 is a potent inducer of apoptosis and that its activity is facilitated by caspase-8 activation, Bid cleavage, Bax translocation to mitochondria, release of cytochrome c into cytoplasm, and subsequently caspase-3 activation, which offers a potential mechanism for the apoptosis-inducing activity of saucernetin-7.

Determination of itopride in human plasma by liquid chromatography coupled to tandem mass spectrometric detection: application to a bioequivalence study.

A simple method using a one-step liquid-liquid extraction (LLE) with butyl acetate followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of itopride in human plasma, using sulpiride as an internal standard (IS). Acquisition was performed in multiple reaction monitoring (MRM) mode, by monitoring the transitions: m/z 359.5>166.1 for itopride and m/z 342.3>111.6 for IS, respectively. Analytes were chromatographed on an YMC C18 reverse-phase chromatographic column by isocratic elution with 1 mM ammonium acetate buffer-methanol (20: 80, v/v; pH 4.0 adjusted with acetic acid). Results were linear (r2=0.9999) over the studied range (0.5-1000 ng mL(-1)) with a total analysis time per run of 2 min for LC-MS/MS. The developed method was validated and successfully applied to bioequivalence studies of itopride hydrochloride in healthy male volunteers.

Effect of ginseng polysaccharide on the stability of lactic acid bacteria during freeze-drying process and storage.

Lactic acid bacteria (LAB) quickly attenuate or are killed during the freeze-drying process and storage. The effect of some natural polysaccharides, which are known as potent antitumor and immunomodulating substances, on the viability of the LAB, Lactobacillus acidophilus and Bifidobacterium breve, on freeze-drying and storage were investigated. Among the polysaccharides tested, red ginseng polysaccharide (RGP) and chitosan significantly inhibited the cell death of the LAB during freeze-drying, and fucoidan and RGP most potently protected the cell death of the LAB during storage. The stabilities of the LAB on the addition of RGP and fucoidan were comparable to that of skimmed milk. However, white ginseng polysaccharide (WGP) did not promote storage stability. When 5% skimmed milk/5% RGP treated LAB were freeze-dried and stored, their viabilities were found to be significantly higher those treated with 5% or 10% RGP. The stabilizing effect of 5% RGP/5% skimmed milk during LAB freeze-drying and storage stability was comparable to that of treatment with 10% skimmed milk. Based on these findings, we believe that RGP beneficially improves the stability of LAB during the freeze-dry process and storage.

Preventive effect of Ginkgo biloba extract (GBB) on the lipopolysaccharide-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 via suppression of nuclear factor-kappaB in RAW 264.7 cells.

During our ongoing efforts to identify bioactive natural products with anti-inflammatory activity, we produced an extract from Ginkgo biloba (GBB) which contains higher levels of the active principles terpene and biflavonoid than EGb, the standard commercially available extract. In the present study, we examined and compared the effects of these two extracts on lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production by the RAW 264.7 macrophage cell line. Our data indicate that GBB is a more potent inhibitor of NO and PGE2 production than EGb 761, and it also significantly decreased tumor necrosis factor (TNF)-alpha release. Consistent with these observations, the protein and mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were found to be inhibited by GBB in a dose-dependent manner. Furthermore, GBB inhibited the LPS-induced DNA binding activity of nuclear factor-kappaB (NF-kappaB), which was associated with the prevention of IkappaB degradation, and subsequently with decreased p65 protein level in the nucleus. These results suggest that GBB inhibits LPS-induced iNOS, COX-2 and TNF-alpha expressions through the down-regulation of NF-kappaB-DNA binding activity.

Saucernetin-8 isolated from Saururus chinensis induced the differentiation of human acute promyelocytic leukemia HL-60 cells.

The present work was performed to investigate the effects of saucernetin-8 on proliferation and differentiation of human leukemia HL-60 cells as well as the underlying mechanisms for these effects. Saucernetin-8 exhibited a potent antiproliferative activity against HL-60 cells. This compound was also found to be a potent inducer of differentiation in human leukemia derived HL-60 cells through the examination of differentiation markers, as assessed by nitroblue tetrazolium reduction test, esterase activity assay, phagocytic activity assay, morphology change, and expression of CD14 and CD66b surface antigens. These results suggest that saucernetin-8 induces the differentiation of human leukemia cells to granulocytes and monocytes/macrophages lineage. Moreover, DNA flow-cytometry indicated that saucernetin-8 induced a G1 phase arrest of HL-60 cells. The protein and mRNA expression levels of p21 were up-regulated during saucernetin-8-dependent HL-60 cell differentiation, whereas the level of c-myc was down-regulated. Taken together, our results suggest that saucernetin-8 may have potential as a therapeutic agent in human leukemia.