The purpose of this study was to examine the effect of a continuous compressive force on rat bone marrow osteoblast-like cells in vitro. Approximately 5.0 × 10⁴ cells were seeded into 35 mm dishes and cultured for 24 h. Thereafter a 2.5 × 2.5 × 0.5 cm glass (which would load the cells with approximately 0.9 g/cm²) was placed on the cell layer for 12 h, and then a glass was removed and continued culturing up to 3 days. Cells cultured without loading were used as a control. The cells were observed using both phase contrast microscopy through the transparent glass and scanning electron microscopy after fixing the cells. Quantitative RT-PCR assays for osteopontin mRNA and osteocalcin mRNA were conducted, and alkaline phosphatase activity was also evaluated. The compressed cells were oriented in mesh-like patterns, and were polygonal and flattened in shape. The compressed cells had a significant down-regulation of osteocalcin mRNA, while there were no significant differences in alkaline phosphatase activity or in osteopontin mRNA between the control and the experimental groups. These results suggest that continuous compression of the cells delays the final cell differentiation of the rat bone marrow cells to active osteoblasts.

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In the present communication, the neuropharmacokinetics of valproate in pentylenetetrazol-kindled epileptogenesis rat hippocampus have been examined by micordialysis. It was found that the maximum concentration and the area under the hippocampus concentration-time curve of valproate in pathological rats were significantly higher than those in the control group. Time to maximum concentration of valproate appeared at 45 min after drug administration, and then the concentration of valproate gradually declined. The results suggest that the pathological damages of rat brain induced by pentylenetetrazol may result in the increase of the valproate level in epileptic rat hippocampus, and the neuropharmacokinetic research of valproate in a chronic kindled conscious animal model with acute administration may help the clinic in the pharmacokinetic optimization of the valproate dosage schedule in epileptic patients.

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The aim of the present study was to investigate the effect of lipopolysaccharide (LPS) and (−)-epigallocatechin-3-gallate (EGCg) on motility of jejunal segments isolated from mice. Endotoxemia, induced by intraperitoneal injection of 10 mg/kg LPS, evoked a significant decrease in the amplitude of spontaneous isometric contractions and an increase in the papaverine-induced relaxations. EGCg induced a dose-dependent relaxation and a decrease in the amplitude and the rate of spontaneous contractions. The effect was observed at relatively low concentration of EGCg (0.125 mM) and was not inhibited by naloxone, phentolamine, or yohimbine. The response to acetylcholine in the presence of EGCg was bigger in endotoxemic animals. Naloxone enhanced the acetylcholine-induced contractions in the presence of EGCg in saline-treated animals, while in endotoxemic animals it was not effective. In conclusion, endotoxemia induces an impairment of spontaneous jejunal contractions recorded in vitro. The EGCg-induced relaxations are smaller in endotoxemic tissues and do not depend on alpha adrenergic and opioid receptors. Additionally, an EGCg-induced increase in the acetylcholine-evoked contractions in endotoxemic animals suggests that EGCg may have a positive effect on LPS-induced impairment in intestinal motility.

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Mammalian cells often use the highly conserved mitogen-activated protein kinase (MAPK) or extracellular signal regulated protein kinase (ERK) cascades to transmit intracellular instructions. These signaling pathways have been proposed to regulate a diverse range of biological functions including apoptosis. Since 2-methoxyestradiol (2ME) and prostaglandin A₂ (PGA₂) play an active role in the induction of apoptosis, the influence of 1 μM 2ME or 20 μg/ml PGA₂ was investigated on ERK1/2 expression levels in three cancer cell lines. PGA₂ exposure led to a statistically significant increase in ERK1/2 expression levels of HeLa and WHCO3 cells. In contrast to the HeLa and WHCO3 cancer cell lines, no effect on ERK1/2 expression levels was observed after exposure of PGA₂ to MCF-7 cells. 2ME caused a statistically significant increase in ERK1/2 expression levels in HeLa, MCF-7 and WHCO3 cells. WHCO3 cells were shown to be more susceptible to the effects of PGA₂ and 2ME compared to the other two cancer cell lines. Since the characteristics of both PGA₂ and 2ME render them as possible anti-tumor agents when compared to conventional chemotherapeutic treatments, understanding the functional role of signaling events and their regulation by interfering pathways after exposure of cells to PGA₂ and 2ME respectively, will provide new insights into mechanisms involved in malignant cell proliferation.

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Human growth hormone_1—43 (hGH_1—43) is an amino terminal fragment of 22 kilodalton hGH (hGH_1—191). In the present study, we developed a specific sandwich-type enzyme linked immunosorbent assay (ELISA) of hGH_1—43. The minimal detectable dose was 3.5 × 10⁻⁴ pmol/well. hGH_1—191 did not show any crossreactivity to hGH_1—43 in the assay up to the dose of 0.25 pmol/well. Using this assay, we investigated the quantitative relationship between hGH_1—191 and hGH_1—43 in contents of normal human pituitary gland and hGH-producing adenoma cells. We also studied the release of hGH_1—191 and hGH_1—43 from cultured hGH-producing pituitary adenoma cells. Means (±SD) ratios of hGH_1—43/hGH_1—191 in the cytosolic fractions were 0.02 ± 0.01% in the normal pituitary and 0.07 ± 0.04% in hGH-producing adenoma cells, respectively. In static experiments, the ratio of hGH_1—43/hGH_1—191 was 1.21 ± 0.50% in the culture medium of hGH-producing pituitary adenoma cells. hGH_1—191 and hGH_1—43 were released in parallel in response to TRH, and the hGH_1—43/hGH_1—191 ratio was 1.37% in perifusion experiments. These observations suggest that the hGH_1—43/hGH_1—191 ratio in cell content is much lower than previously reported. It was also suggested that the ratio of hGH_1—43/hGH_1—191 is higher in the releasable pool of hGH in the pituitary and hGH_1—43 might be secreted in a similar fashion to hGH_1—191.

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Calcitonin gene-related peptide (CGRP) is a neuropeptide, which has multiple functions, and CGRP-containing nerves are prolific in the periodontal tissues. Tooth movement accompanies active tissue remodeling, including bone formation and resorption in the periodontal ligament. Although it has been suggested that CGRP contributes to tissue remodeling incident to tooth movement, little information is available for this issue. In the present study, immunohistochemistry combined with a computer-assisted quantitative analysis was employed to examine chronological changes in staining intensity for CGRP in osteoblasts during experimental tooth movement. The maxillary canine on one side was moved distally and fixed for 1 h to 56 days, leaving an untreated canine at the opposite side to serve as a control. Undecalcified sagittal sections of each maxilla, 6 μm in thickness, were processed by immunohistochemistry for CGRP. The distal alveolar bone around the root apex was selected for the analysis because of active bone formation in this area, due to constant tension during tooth movement. Percent staining intensity (SI) for osteoblastic CGRP in the experimental and control groups was calculated using a Zeiss microphotometer equipped with a Microscope Analysis with Photometric Scanning (MAPS) program. The mean SI for CGRP increased daily, peaked at postoperative Day 7, and decreased gradually thereafter. The relative ratio of SI in the experimental and control groups was about 200% at 1 h postoperatively and increased to approximately 300% during postoperative Days 7—14. The surface area of CGRP-labeled osteoblasts also showed the same alternation pattern as that of SI. These findings indicate that CGRP is released from neurons and can be bound to osteoblasts, and that orthodontic forces can alter the levels of SI for CGRP, suggesting the involvement of CGRP in regulation of osteoblastic activity. High SI for CGRP in the osteoblasts at time periods when the surface area of osteoblasts was increased, suggests that CGRP possibly participates in mediating the effects of orthodontic forces on alveolar osteoblasts.

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Src-suppressed C kinase substrate (SSeCKS) is expressed in a variety of cells and plays an important role in cell differentiation, migration and shaping via remodeling of the actin-based cytoskeleton. In the present study, the cellular distribution of SSeCKS was studied in the peripheral nervous system and sensory organs of mice by in situ hybridization and immunohistochemistry. In the dorsal root ganglia and trigeminal ganglia, small- and medium-sized neurons expressed SSeCKS, whereas large-sized neurons showed negative or faintly positive reactions. Definite positive signals of SSeCKS mRNA and protein in the sensory ganglia were also found in some satellite cells which frequently enveloped ganglion cells without SSeCKS expression. In autonomic ganglia (celiac ganglia, pelvic ganglia, and intestinal myenteric nerve plexus), SSeCKS immunoreactivity was confined to satellite cells, and not detectable in neuronal somata. No significant immunoreactivity for SSeCKS appeared in most sensory organs, including the retina, organ of Corti, taste buds, and olfactory mucosa. In contrast, the vomeronasal organ displayed intense and selective expression of SSeCKS in the sensory epithelium, where only supporting cells were immunolabeled. SSeCKS-immunoreactive Schwann cells were widely distributed in nerve bundles including the vomeronasal nerve, olfactory nerve, and dorsal root. These findings suggest that SSeCKS might be involved in regulation and homeostasis of the peripheral nervous system and of the vomeronasal organ among sensory organs.

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