Optimal fluorescent-dye staining time for the real-time detection of microbes: a study of Saccharomyces cerevisiae.

AIMS: To provide information on the time-dependent behaviour of microbe staining by fluorescent dyes in the order of seconds, which is important in terms of the recent rapid and online techniques for microbe measurements and/or environmental microbe analysis. METHODS AND RESULTS: For combinations of yeast (Saccharomyces cerevisiae) and typical dyes, including DAPI (4',6-diamidino-2-phenylindole) and Auramine-O, a suspension of yeast cells in ultrapure water was injected into a dye solution in a micro cuvette placed inside a spectrofluorometer and the fluorescence intensity of the resulting solution was measured at 1 s intervals, starting immediately after the mixing and continued until the time for the maximum intensity using various concentrations of yeast and dyes. The relaxation time τ, which corresponds to ~63·2% of the maximum fluorescence intensity, was shown to decrease to below 1 s with increasing DAPI concentration, whereas it remained constant for 2-3 s with increasing Auramine-O concentration, for example at a yeast concentration of 100 µg ml-1 . CONCLUSIONS: For the conditions of yeast >10 µg ml-1 , DAPI >1 µg ml-1 and Auramine-O >0·1 µg ml-1 , τ could be adjusted to below 5 s to achieve a rapid and stable staining. SIGNIFICANCE AND IMPACT OF THE STUDY: Design and operating conditions for rapid and online measurements of microbes can be optimized.

Cell dynamics in Hertwig's epithelial root sheath and surrounding mesenchyme in mice irradiated to the head.

OBJECTIVE: To investigate the mechanisms that cause damage to root formation as a result of irradiation to the mouse head, morphological changes in molar dental roots and cell dynamics in Hertwig's epithelial root sheath (HERS), and surrounding mesenchymal tissue were examined. MATERIALS AND METHODS: To perform the experiments, 5-day-old C57BL/6 mice were randomly divided into three groups: the control group (0 Gy) and irradiated groups (10 and 20 Gy). Micro-CT analysis, HE staining, immunohistochemistry analysis, and TUNEL assay were then performed. RESULTS: Roots in irradiated mice were dose-dependently shorter than those of control mice. Cells located outside the root dentin, with abnormal morphology in irradiated mice, were positive for an odontoblast marker. HERS fragmentation occurred earlier in irradiated mice than in control mice, and HERS was trapped by the calcified apical tissue. A dose-dependent reduction in the number of proliferating cells within the apical dental pulp and periapical periodontal ligament surrounding HERS was observed in irradiated mice. Apoptotic cells in the dental pulp and periodontal ligament surrounding HERS were hardly seen. CONCLUSIONS: These results indicate that the early disappearance of HERS and the proliferative suppression of the surrounding mesenchymal cells, which was induced by irradiation, caused dental root malformation.

Magnetic separation and characterization of keratinocyte stem cells from human gingiva.

BACKGROUND AND OBJECTIVE: Although keratinocyte stem cells play a key role in tissue homeostasis, wound healing and neoplasia, they remain difficult to identify and characterize. The purpose of this study was to isolate and characterize an oral keratinocyte stem-cell population. MATERIAL AND METHODS: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using α(6) ß(4) integrin and a proliferation-related marker, CD71. The isolated cell fractions were analyzed for cell size, cell cycle stage (using flow cytometry) and colony-forming ability. The expression of stem cell markers p63 and cytokeratin 19 and of differentiation markers cytokeratin 10 and involucrin was checked using immunocytochemical analysis. RESULTS: The stem cell CD71(neg) fraction had the smallest cell size compared with CD71(pos) and fractions [780.7 ± 141.5 (pixels), 1422.9 ± 264.6 (pixels) and 3844.4 ± 220.1 (pixels) respectively, p < 0.01; analysis of variance (ANOVA)]. Also, the CD71(neg) subpopulation consistently had the highest colony-forming ability among the three cell fractions (126.2 ± 21.7 vs. 32.8 ± 4.5 vs. 12.4 ± 2.1 compared with CD71(pos) and subpopulations, respectively, p < 0.01; ANOVA). Moreover, the CD71(neg) fraction contained more quiescent cells and fewer actively cycling cells than the CD71(pos) cell fraction. The candidate stem cells were positive for cytokeratin 19 and p63 keratinocyte stem cell markers, while differentiation markers such as cytokeratin 10 or involucrin were absent. CONCLUSION: The human gingival CD71(neg) cell fraction, separated by a magnetic system, demonstrated several characteristics of gingival keratinocyte stem cells. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.

Maillard reaction, mitochondria and oxidative stress: potential role of antioxidants.

Glycation and oxidative stress are two important processes known to play a key role in complications of many disease processes. Oxidative stress, either via increasing reactive oxygen species (ROS), or by depleting the antioxidants may modulate the genesis of early glycated proteins in vivo. Maillard Reactions, occur in vivo as well as in vitro and are associated with the chronic complications of diabetes, aging and age-related diseases. Hyperglycaemia causes the autoxidation of glucose, glycation of proteins, and the activation of polyol metabolism. These changes facilitate the generation of reactive oxygen species and decrease the activity of antioxidant enzymes such as Cu,Zn-superoxide dismutase, resulting in a remarkable increase of oxidative stress. A large body of evidence indicates that mitochondria alteration is involved and plays a central role in various oxidative stress-related diseases. The damaged mitochondria produce more ROS (increase oxidative stress) and less ATP (cellular energy) than normal mitochondria. As they are damaged, they cannot burn or use glucose or lipid and cannot provide cell with ATP. Further, glucose, amino acids and lipid will not be correctly used and will accumulate outside the mitochondria; they will undergo more glycation (as observed in diabetes, obesity, HIV infection and lipodystrophia). The objective of this paper is to discuss how to stop the vicious circle established between oxidative stress, Maillard Reaction and mitochondria. The potential application of some antioxidants to reduce glycation phenomenon and to increase the antioxidant defence system by targeting mitochondria will be discussed. Food and pharmaceutical companies share the same challenge, they must act now, urgently and energetically.

Scanning electron microscope imaging of bacteria based on DNA sequence.

AIMS: To develop a scanning electron microscopic approach using in situ hybridization (SEM-ISH) for gaining both genetic and morphological information about target bacteria. METHODS AND RESULTS: Target cells were hybridized with DNA-targeted polynucleotide probes, and a tyramide signal amplification system was used to increase the sensitivity. The protocol of SEM-ISH enabled to detect low copy number target DNA sequences in individual cells. CONCLUSIONS: SEM-ISH allowed the in situ detection of bacteria carrying a specific gene. SIGNIFICANCE AND IMPACT OF THE STUDY: Combining morphological study with SEM and ISH techniques appears to be a valuable tool to understand the spatial distribution of target cells in complex microbial communities on various materials.

Heat island mitigation using water retentive pavement sprinkled with reclaimed wastewater.

In Japan, reclaimed wastewater has been recycled widely for non-potable urban applications and it is to be used for sprinkling roads to mitigate heat island in urban areas. To assess the heat island mitigation effects of the sprinkling reclaimed wastewater on water retentive pavement, we carried out a survey at Shiodome-District, Tokyo. The temperatures of air and roads, humidity, and WBGT (Wet-bulb globe temperature) were measured and heat flux was estimated to compare the condition of the areas with/without sprinkling. The following results were obtained. 1) Sprinkling reclaimed wastewater decreased the road surface temperature by 8 degrees during the daytime and by 3 degrees at night: temperatures equal to those on planting zones. Nevertheless sprinkling was done only in the daytime, the temperature decrease effect was not only obtained during the daytime: it continued through the night, due to the water retentive pavement. 2) Sprinkling reclaimed wastewater reduced the amount of sensible heat flux and increased that of latent heat flux. These results suggest that sprinkling reclaimed wastewater on water retentive pavement can effectively mitigate the heat island phenomenon.

Somatostatin inhibits the excitability of rat small-diameter trigeminal ganglion neurons that innervate nasal mucosa and project to the upper cervical dorsal horn via activation of somatostatin 2a receptor.

This study investigated whether somatostatin (SST) modulates the excitability of nociceptive trigeminal ganglion (TRG) neurons that innervate the nasal mucosa and project to the upper cervical (C(1)) dorsal horn by using perforated-patch clamping, retrograde-labeling, and immunohistochemistry. Fluorogold (FG) retrograde labeling was used to identify the rat TRG neurons innervating the nasal mucosa, while microbeads (MB) were used to label neurons projected onto the superficial layer of the C(1) dorsal horn. FG-labeled small-diameter TRG neurons exhibited SST(2A) receptor immunoreactivity (19%) and half of these neurons were also labeled with MB. In whole-cell current-clamp mode, most (72%) of the dissociated FG-/MB-labeled TRG neurons were hyperpolarized by application of SST. The hyperpolarization was evoked by SST in a concentration-dependent manner (0.1-10 microM) and the responses were associated with a decrease in the cell input resistance. The minimum concentration to elicit a significant hyperpolarization was 1 microM. The repetitive firings during a depolarizing pulse were significantly reduced by SST (1 microM) application. The hyperpolarization and decreased firing evoked by SST were both blocked by the SST(2) receptor antagonist, CYN154806 (1 microM). Under voltage-clamp conditions, SST (1 microM) significantly increased the voltage-gated K(+) transient (I(A)) and sustained (I(K)) currents and these increases were abolished by coapplication of CYN154806 (1 microM). In the presence of both 4-aminopyridine (6 mM) and tetraethylammonium (10 mM), no significant changes in the membrane potential in response to SST application were found. These results suggest that modulation of trigeminal nociceptive transmission in the C(1) dorsal horn by activation of SST(2A) receptors occurs at the level of small-diameter TRG cell bodies and/or their afferent terminals, and that this may be related to regulation of protective upper-airway reflexes.

Enhanced excitability of rat trigeminal root ganglion neurons via decrease in A-type potassium currents following temporomandibular joint inflammation.

The aim of the present study was to investigate the effect of temporomandibular joint inflammation on the excitability of trigeminal root ganglion neurons innervating the temporomandibular joint using a perforated patch-clamp technique. Inflammation was induced by injection of complete Freund's adjuvant into the rat temporomandibular joint. The threshold for escape from mechanical stimulation in the temporomandibular joint-inflamed rats was significantly lower than that in control rats. Fluorogold labeling was used to identify the trigeminal root ganglion neurons innervating the site of inflammation. When voltage-clamp (V(h)=-60 mV) conditions were applied to these Fluorogold-labeled small diameter trigeminal root ganglion neurons (<30 mum), voltage-dependent transient K(+) current densities were significantly reduced in the inflamed rats compared with controls. In addition, the voltage-dependence of inactivation of the voltage-dependent transient K(+) current was negatively shifted in the labeled temporomandibular joint-inflamed trigeminal root ganglion neurons. Furthermore, temporomandibular joint inflammation significantly reduced the threshold current and significantly increased action potential firings evoked at two-fold threshold in the Fluorogold-labeled small trigeminal root ganglion neurons. Application of 4-aminopyridine (0.5mM) to control trigeminal root ganglion neurons mimicked the changes in the firing properties observed after complete Freund's adjuvant treatment. Together, these results suggest that temporomandibular joint inflammation increases the excitability of trigeminal root ganglion neurons innervating temporomandibular joint by suppressing voltage-dependent transient K(+) current via a leftward shift in the inactivation curve. These changes may contribute to trigeminal inflammatory allodynia in temporomandibular joint disorder.

Development of Helicobacter pylori-induced gastric carcinoma in Mongolian gerbils.

Helicobacter pylori is classified by IARC/WHO as a definite human gastric carcinogen, despite "inadequate experimental evidence." To obtain direct evidence concerning this relationship, we investigated the histopathological findings of gastric mucosa using a model of H. pylori infection in Mongolian gerbils. The animals were challenged p.o. with H. pylori ATCC-43504 and sacrificed at 6, 12, and 18 months after inoculation for histological examination. All inoculated animals were infected with H. pylori. Severe infiltration of the lamina propria by polymorphonuclear and mononuclear cells appeared in the lesser curvature of the antrum, with an increase in epithelial cell proliferation, and the infiltration extended to the body. Atrophic gastritis and focal intestinal metaplasia also appeared in the lesser curvature of the antral mucosa at 6 months after inoculation. Intestinal metaplasia became severe, with dysplasia, after that. At 18 months after H. pylori inoculation, two of five infected animals showed three well-differentiated gastric cancers. The uninfected control animals showed no abnormal findings throughout the entire observation period. Here, it was confirmed that H. pylori infection alone causes gastric cancer in an animal model.

Flow capacity of internal mammary artery grafts: early restriction and later improvement assessed by Doppler guide wire. Comparison with saphenous vein grafts.

OBJECTIVES: The purpose of this study was to assess flow dynamics and flow capacities of internal mammary artery and saphenous vein grafts to the left anterior descending coronary artery. BACKGROUND: The postoperative flow capacity of internal mammary artery grafts to the left anterior descending coronary artery has been reported to be restricted compared with that of saphenous vein grafts in studies using radionuclide angiography. A recently developed Doppler guide wire has been used to analyze the flow dynamics of bypass grafts and to clarify the mechanism of this limited flow capacity. METHODS: Phasic flow velocity recordings were obtained in the midportion of the bypass graft and within the native left anterior descending artery, using a 0.018-in. (0.046-cm) 12-MHz Doppler guide wire, in 53 patients: 27 patients with an internal mammary artery graft (16 with a new graft assessed 1 month postoperatively and 11 with an old graft assessed at 1 year) and 26 patients with a saphenous vein graft (13 with a new graft assessed 1 month postoperatively and 13 with an old graft assessed at 1 year). All patients were studied at baseline rest and during hyperemia induced by intravenous infusion of dipyridamole, 0.56 mg/kg body weight, over 4 min. RESULTS: In the left anterior descending artery itself, systolic and diastolic peak velocities, the time average of the instantaneous spectral peak velocity (time-averaged peak velocity), vessel diameter and the calculated flow volume did not differ significantly among the four graft groups. The time-averaged peak velocity was significantly greater for new than for old arterial grafts or for new or old vein grafts (mean +/- SD 27 +/- 9 vs. 19 +/- 6, 11 +/- 5 and 12 +/- 6 cm/s, respectively, p < 0.01). However, because the diameter of new arterial grafts was significantly smaller than that of the other three grafts (2.4 +/- 0.1 vs. 2.9 +/- 0.2 [p < 0.05], 3.6 +/- 0.6 [p < 0.01] and 3.4 +/- 0.5 mm [p < 0.01], respectively), there was no difference in calculated flow volumes at rest (62 +/- 17 vs. 58 +/- 15, 61 +/- 18 and 58 +/- 19 ml/min, respectively, p = NS) between new arterial grafts and the other grafts. Although the maximal time-averaged peak velocity during hyperemia was significantly greater in new than in old arterial grafts or new or old vein grafts (47 +/- 17 vs. 40 +/- 7, 31 +/- 8 and 34 +/- 12 cm/s, respectively, p < 0.01), the flow reserve of new arterial grafts was significantly smaller than that of the other three groups (1.8 +/- 0.3 vs. 2.6 +/- 0.3, 2.8 +/- 0.5 and 3.0 +/- 0.6, respectively, p < 0.01) because the baseline time-averaged peak velocity of these new grafts was far greater than that of the other groups. CONCLUSIONS: Internal mammary artery graft flow early after operation is characterized by a higher rest velocity than that of vein graft flow. This high velocity maintains flow volume at baseline condition in compensation for the smaller diameter. Although flow reserve does not differ significantly between new and old vein grafts, that for internal mammary artery grafts is significantly reduced soon after bypass surgery. This restricted flow capacity improves late postoperatively because of an increase in diameter and a decrease in flow velocity from baseline levels.

Flow dynamics of angiographically no-flow patent internal mammary artery grafts.

OBJECTIVES: This study sought to assess the flow dynamics of internal mammary artery grafts (IMAGs) in no-flow situations by use of a Doppler guide wire. BACKGROUND: Functionally no-flow and anatomically patent IMAGs have been reported by angiography in patients with a patent recipient coronary artery. METHODS: The study included 12 patients with an IMAG to the left anterior descending coronary artery (LAD) in whom no-flow patency of the graft was suspected angiographically. Thirteen patients with a normally functioning IMAG whose LAD was occluded in the proximal portion and was supplied only from the graft served as control patients. Phasic flow velocities were recorded in the distal portion of the graft and the recipient LAD using a 0.014-in., 15-MHz Doppler guide wire at rest and during hyperemia (0.14-mg/kg body weight per min intravenous adenosine infusion). RESULTS: There were no significant differences in systolic (15+/-3 vs. 19+/-6 cm/s, p = NS), diastolic (35+/-11 vs. 37+/-7 cm/s, p = NS) and time-averaged peak velocities at rest (20+/-5 vs. 21+/-5 cm/s, p = NS), during hyperemia (51+/-12 vs. 54+/-8 cm/s, p = NS) and in coronary flow velocity reserve (2.8+/-0.9 vs. 2.7+/-0.3, NS) in the native LAD in patients with a no-flow patent graft versus control patients. Within the graft, to and fro signals with systolic reversal and diastolic anterograde flow were seen in the no-flow patent grafts, although anterograde flow signals were recorded in systole and diastole in control patients. Systolic (-28+/-19 vs. 22+/-9 cm/s, p < 0.01), diastolic (18+/-17 vs. 44+/-14 cm/s, p < 0.01) and time-averaged (-2+/-6 vs. 26+/-9 cm/s, p < 0.01) peak velocities at rest were significantly smaller in the no-flow patent grafts than in control grafts. During hyperemia, anterograde flow became predominant, with a reduction in retrograde systolic flow signal and an increase in diastolic flow velocity and time-averaged peak velocity in the no-flow patent grafts, and no-flow situations disappeared temporarily. CONCLUSIONS: Functionally no-flow situations of IMAGs manifesting to and fro signals with systolic flow reversal and diastolic antegrade low flow velocity are temporary conditions in certain hemodynamic circumstances, and these grafts function as conduits during hyperemic states.

Real-time PCR assays based on distinct genomic regions for cytomegalovirus reactivation following hematopoietic stem cell transplantation.

Real-time PCR has many advantages compared with antigenemia and qualitative PCR assays for detecting cytomegalovirus (CMV) infection in patients following SCT. However, the procedure used in each report was not standardized. This study compares the CMV load detected by real-time PCR assays amplifying distinct genomic regions. Real-time PCR assays based on US17, UL65, immediate early protein (IE) and glycoprotein B(gB) were selected and comparisons were made between each genomic region, and with antigenemia and nested PCR (IE region) in 18 SCT patients. The CMV load detected by real-time PCR using all combinations of primers targeting distinct genomic regions and by antigenemia assays correlated well. However, US17 and UL65-PCR could detect CMV earlier than gB-PCR, antigenemia and nested PCR assays. In longitudinal analysis, gB-PCR demonstrated a trend for showing a lower viral load in some patients than US17-, UL65- and IE-PCR. Moreover, the results suggest that a cutoff level of 500 copies/ml might be used to decide when to initiate treatment. We propose that monitoring should be carried out using real-time PCR assays targeting the US17 region and that a CMV load of 500 copies/ml could be used as a cutoff value for initiating treatment in patients following SCT, receiving immunoglobulin prophylaxis.

High plasma concentrations of osteopontin in patients with interstitial pneumonia.

Osteopontin (OPN) produced by alveolar macrophages functions as a fibrogenic cytokine in the development of bleomycin (BLM)-induced murine pulmonary fibrosis, and OPN mRNA is expressed on lung tissues from patients with idiopathic pulmonary fibrosis (IPF). The present study investigates plasma OPN levels in human interstitial pneumonia (IP) and their relationships with disease severity by analyzing the correlation between plasma OPN concentrations and pulmonary functions. The concentrations of OPN in plasma were measured in 17 patients with IP, in 9 with sarcoidosis and in 20 healthy controls using an antigen-capture enzyme-linked immunosorbent assay. The concentrations of OPN in plasma were significantly higher in IP patients than in those with sarcoidosis or in controls. Based on a Receiver Operating Characteristic curve analysis, cut-off points between 300 and 380 ng/ml discriminated between IP and control subjects with 100% sensitivity and 100% specificity. In such case, the sensitivity for sarcoidosis decreased (55.5-33.3%) in cut-offs with 100% specificity. Plasma OPN levels inversely and closely correlated with arterial oxygen tension (PaO2) in patients with IP. Immunohistochemically, OPN was localized predominantly in macrophages and airway epithelium. These findings suggest that plasma OPN levels were found to be associated with the presence of IP, and that OPN play an important role in the development of IP.

Activation of GABA(B) receptors potentiates inward rectifying potassium currents in satellite glial cells from rat trigeminal ganglia: in vivo patch-clamp analysis.

In a previous study, we demonstrated that inflammation suppressed inward rectifying K(+) (Kir) currents in satellite glial cells (SGCs) from the trigeminal ganglia (TRGs) and that this impairment of glial potassium homeostasis in the trigeminal ganglion (TRG) contributed to trigeminal pain. The aim of the present study was to investigate whether activation of GABAB receptors modulates the Kir current in SGCs using in vivo patch-clamp and immunohistochemical techniques. Immunohistochemically, we found that immunoreactivity for glial-specific Kir channel subunit Kir4.1 and the GABAB receptor was co-expressed in SGCs from the TRGs. In vivo whole-cell recordings were made using SGCs from the TRGs of urethane-anesthetized rats. Application of baclofen, a GABAB receptor agonist, significantly increased the mean peak amplitude of Kir currents in a concentration-dependent and reversible manner. Baclofen-induced potentiation of the Kir current was abolished by co-application of 3-amino-2-(4-chlorophenyl)-2-hydroxyprophylsulfonic acid (saclofen). In addition, baclofen significantly potentiated the density of the Ba(2+)-sensitive Kir current, and resulted in hyperpolarization of the mean membrane potential. These results suggest that activation of GABAB receptors potentiates the Kir current in SGCs and that GABA released from the TRG neuronal soma could contribute to buffering of extracellular K(+) concentrations following excitation of TRG neurons during the processing of sensory information, including the transmission of noxious stimuli.

Ethanol has thyrotropin-like activity in cultured porcine thyroid follicles.

We describe TSH-like activity of ethanol for thyroid hormone formation in the physiological culture system. Porcine thyroid follicles were preincubated with 0-100 mM (0-0.58%) ethanol in the presence of 0-1280 microU/ml bovine TSH for 24 h; these follicles were then incubated with the mixture of Na125I and NaI to measure iodide uptake, iodine organification, and de novo thyroid hormone formation. Ethanol stimulated iodide uptake in a dose-response manner in TSH-free medium. Ethanol augmented the effect of TSH on iodide uptake, iodide organification, and thyroid hormone formation in the presence of 20-80 microU/ml TSH. When TSH concentration was 320 microU/ml or greater, ethanol no longer stimulated iodide uptake and thyroid hormone formation. Ethanol mediated iodide uptake and iodine organification were inhibited by potassium perchlorate and propylthiouracil respectively. The effect of ethanol on the thyroid follicle was reversible 24 h after removal of ethanol from the medium. The mechanism of TSH-like activity of ethanol was studied by measuring cAMP generation and Na+K+ATPase activity, a sodium pump necessary for iodide transport, in the presence of 0-1280 microU/ml TSH. Ethanol increased cAMP production in TSH-free medium; the increment of cAMP by ethanol was more prominent when 20-80 microU/ml TSH were present. Ethanol also augmented (Bu)2cAMP-mediated iodide uptake and TSH-mediated thyroid Na+K+ATPase activity. Thus, TSH-like activity of ethanol for thyroid hormone formation can be explained by activation of the cAMP system and Na+K+ATPase activity. Our results indicate that ethanol concentrations equivalent to the blood level of moderate to heavy alcohol drinkers exert TSH-like activity in the thyroid follicle.

Estrogen blocks parathyroid hormone (PTH)-stimulated osteoclast-like cell formation by selectively affecting PTH-responsive cyclic adenosine monophosphate pathway.

Several lines of evidence have previously indicated that estrogen inhibits PTH-induced bone resorption in vivo and in vitro. However, its mechanism remains unknown. Therefore, the present study was performed to investigate the effect of estrogen on PTH-stimulated osteoclast-like cell formation and clarify its mechanism. 17 beta-estradiol (17 beta-E2) significantly antagonized osteoclast-like cell formation stimulated by 10(-8) M human (h) PTH-(1-34) as well as 10(-8) M hPTH-related peptide (PTHrP)-(1-34)in osteoblast-containing mouse bone cell cultures. The conditioned medium derived from osteoblastic SaOS-2 cells or MC3T3-E1 cells pretreated with both PTH-(1-34) (10(-8)M) and 17 beta-E2(10(-8)M) stimulated osteoclast-like cell formation from hemopoietic blast cells more weakly than conditioned medium from cells pretreated with PTH-(1-34) alone. Moreover, 10(-8) M 17 beta-E2 significantly blocked the formation of osteoclast-like cells stimulated by 10(-8) M hPTH-(1-34) in spleen cell cultures derived from 5-fluorouracil-pretreated mice. On the other hand, 10(-8) M 17 beta-E2 significantly inhibited osteoclast-like cell formation stimulated by dbcAMP (10(-4)M) and Sp-cAMPS (10(-4)M), as well as forskolin (10(-5)M) in mouse bone cell cultures. In contrast, 10(-8)M 17 beta-E2 did not affect PMA (10(-7)M)-, A23187 (10(-7)M)-, or BAYK-8644 (5 x 10(-6) M)-stimulated osteoclast-like cell formation. In conclusion, the present study demonstrated that estrogen inhibits PTH-stimulated osteoclast-like cell formation by directly acting on hemopoietic blast cells as well as by indirectly acting on them via osteoblasts. The inhibitory effects of estrogen on PTH-stimulated osteoclast-like cell formation seemed to be mediated through blocking the cAMP-dependent protein kinase pathway but not by blocking calcium/protein kinase C.

Stimulatory effects of parathyroid hormone and 1,25-dihydroxyvitamin D3 on insulin-like growth factor-binding protein-5 mRNA expression in osteoblastic UMR-106 cells: the difference between transient and continuous treatments.

The transient treatment with parathyroid hormone (PTH) for 12 h, followed by its removal for 36 h, stimulated insulin-like growth factor-binding protein (IGFBP)-5 mRNA expression more strongly than the continuous treatment for 48 h in osteoblastic UMR-106 cells. The transient but not continuous treatment with A23187 also stimulated it. In contrast, 1,25-dihydroxyvitamin D3 stimulated it, irrespective of the treatment design. IGFBP-5 stimulated type-1 procollagen mRNA expression. The present study first indicated that the transient treatment with PTH more effectively stimulated IGFBP-5 mRNA expression than its continuous treatment partly via an increase in intracellular calcium and suggested that IGFBP-5 might be involved in the anabolic action of PTH in bone.

Discovery of novel mesangial cell proliferation inhibitors using a three-dimensional database searching method.

A three-dimensional pharmacophore model of mesangial cell (MC) proliferation inhibitors was generated from a training set of 4-(diethoxyphosphoryl)methyl-N-(3-phenyl-[1,2,4]thiadiazol-5-yl)benzamide, 2, and its derivatives using the Catalyst/HIPHOP software program. On the basis of the in vitro MC proliferation inhibitory activity, a pharmacophore model was generated as seven features consisting of two hydrophobic regions, two hydrophobic aromatic regions, and three hydrogen bond acceptors. Using this model as a three-dimensional query to search the Maybridge database, structurally novel 41 compounds were identified. The evaluation of MC proliferation inhibitory activity using available samples from the 41 identified compounds exhibited over 50% inhibitory activity at the 100 nM range. Interestingly, the newly identified compounds by the 3D database searching method exhibited the reduced inhibition of normal proximal tubular epithelial cell proliferation compared to a training set of compounds.

Opioidergic modulation of excitability of rat trigeminal root ganglion neuron projections to the superficial layer of cervical dorsal horn.

The aim of the present study was to investigate the effect of a micro-opioid receptor agonist DAMGO (Tyr-d-Ala-Gly-NMe-Phe-Gly-ol) on the excitability of trigeminal root ganglion (TRG) neurons, projecting onto the superficial layer of the cervical dorsal horn, by using the perforated-patch technique and to determine whether TRG neurons show the expression of mRNA or functional protein for micro-opioid receptors by using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. TRG neurons projecting onto the superficial layer of the cervical dorsal horn were retrogradely labeled with Fluorogold (FG). The cell diameter of FG-labeled TRG neurons was small (<30 microm). Under voltage-clamp (V(h)=-60 mV), voltage-dependent K(+) currents were recorded in the TRG neurons and isolated by blocking Na(+) and Ca(2+) currents with appropriate ion replacement. Separation of the K(+) current components was achieved by the response to variation in the conditioning voltage. Two distinct K(+) current components, a transient (I(A)) and sustained (I(K)), were identified. DAMGO significantly increased I(A) by 57% (20 microM) and in a dose-dependent manner (1-50 microM). Similarly, I(K) was also enhanced by DAMGO administration (42%, 20 microM). The augmentation of both I(A) and I(K) was antagonized by a micro-opioid receptor antagonist, CTOP (d-Phe-Cys-Thr-d-Trp-Orn-Thr-Pen-Thr-NH(2)). Hyperpolarization of the membrane potential was elicited by DAMGO (20 microM) and the response was associated with a decrease in the input resistance. DAMGO induced hyperpolarization was blocked by CTOP. DAMGO-sensitive I(A) and I(K) currents were antagonized by K(+) channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA). In the presence of both 4-AP and TEA, no significant changes in membrane potential induced by DAMGO application were observed. In the presence of BaCl(2), DAMGO evoked hyperpolarization with decreased resistance was observed. The firing rate of action potentials and the first spike duration induced by depolarizing step pulses were decreased in the presence of DAMGO. RT-PCR analysis demonstrated the expression of mRNA for micro-opioid receptors in the trigeminal ganglia. The micro-opioid receptor immunoreactivity was expressed in the small diameter FG-labeled TRG neurons. These results suggest that the activation of micro-opioid receptors inhibits the excitability of rat small diameter TRG neurons projecting on the superficial layer of the cervical dorsal horn and this inhibition is mediated by potentiation of voltage-dependent K(+) currents. We therefore concluded that modulation of nociceptive transmission in the trigeminal system, resulting in the functional activation of micro-opioid receptors, occurs at the level of small TRG cell bodies and/or their primary afferent terminals, which contribute to opioid analgesia in the trigeminal pain.

Effect of pravastatin (10 mg/day) on progression of coronary atherosclerosis in patients with serum total cholesterol levels from 160 to 220 mg/dl and angiographically documented coronary artery disease. Coronary Artery Regression Study (CARS) Group.

To evaluate the effect of pravastatin on progression of coronary atherosclerosis in normocholesterolemic patients with coronary artery disease (CAD), 90 patients with CAD and serum cholesterol levels of 160 to 220 mg/dl were randomized into a pravastatin (10 mg/day) group (n = 45) and control group (n = 45) in a 2-year study. The proportions of patients with progression (an increase of > or = 15% in percent stenosis) and regression (a decrease of > or = 15% in percent stenosis) of coronary atherosclerosis were compared between the 2 groups. Of 90 patients, 80 (89%) had a final angiogram: the pravastatin (n = 39) and control group (n = 41). Percent changes in total cholesterol, low-density lipoprotein cholesterol, and apoprotein B levels were significantly greater in the pravastatin group than in the control group (total cholesterol -11 +/- 12% vs 3 +/- 15%, p < 0.01; low-density lipoprotein cholesterol -18 +/- 16% vs 4 +/- 21%, p < 0.01; apoprotein B -5 +/- 20% vs 6 +/- 20%, p < 0.05). The proportion of patients with progression of coronary atherosclerosis was significantly smaller in the pravastatin group than in the control group (21% vs 49%, p < 0.05). The proportion of patients with disease regression did not differ in the 2 groups (3% vs 2%, p = NS). In conclusion, this study indicates that cholesterol-lowering therapy with pravastatin can prevent the progression of coronary atherosclerosis even in normocholesterolemic patients with established CAD.