Expression profile of active genes in human periodontal ligament and isolation of PLAP-1, a novel SLRP family gene.

Periodontal ligament (PDL) is one of the most important tissues in maintaining the homeostasis of tooth and tooth-supporting tissue, periodontium. In this study, we investigated the expression profile of active genes in the human PDL obtained by collecting sequences with a 3'-directed cDNA library, which faithfully represents the composition of the mRNA population. We succeeded in obtaining a total of 1752 cDNA sequences by sequencing randomly selected clones and identified a total of 1318 different species as gene signatures (GS) by their sequence identity, 344 of which were known genes in the GenBank, and 974 of which were new genes. The resulting expression profile showed that collagen type I and type III were the most abundant genes and that osteogenesis-related proteins, such as SPARC/osteonectin and osteoblast specific factor 2, were highly expressed. By comparing the expression profile of PDL with 44 profiles similarly obtained with unrelated human cell/tissue, nine novel genes, which are probably expressed specifically in PDL, were discovered. Among them, we cloned a full-length cDNA of GS5096, which is frequently expressed in freshly-isolated periodontal tissue. We found that it encodes a novel protein, which is a new member of the class I small leucine-rich repeat proteoglycan family, and designated it PLAP-1 (periodontal ligament associated protein-1). PLAP-1 mRNA expression was confirmed in in vitro-maintained PDL cells and was enhanced during the course of the cytodifferentiation of the PDL cells into mineralized tissue-forming cells such as osteoblasts and cementoblasts. These findings suggest the involvement of PLAP-1 in the mineralized matrix formation in PDL tissues.

CD28 costimulation is required not only to induce IL-12 receptor but also to render janus kinases/STAT4 responsive to IL-12 stimulation in TCR-triggered T cells.

The activation of resting T cells for the acquisition of various functions depends on whether CD28 costimulatory signals are provided upon T cell receptor stimulation. Here, we investigated how CD28 costimulation functions to allow TCR-triggered resting T cells to acquire IL-12 responsiveness. When T cells are stimulated with low doses of anti-CD3 mAb, CD28 costimulation was required for the optimal levels of IL-12 receptor (IL-12R) expression. However, stimulation of T cells with high doses of anti-CD3 alone induced comparable levels of IL-12R expression to those induced upon CD28 costimulation. Nevertheless, there was a substantial difference in IL-12 responsiveness between these two groups of T cells: compared to anti-CD28-costimulated T cells, T cells that were not costimulated with anti-CD28 exhibited decreased levels of Janus kinases (JAK) JAK2/TYK2 and STAT4 phosphorylation and IFN-y production following IL-12 stimulation. Importantly, STAT6 phosphorylation following IL-4 stimulation was not decreased in anti-CD28-uncostimulated T cells. These resutls indicate that CD28 costimulation not only contributes to up-regulating IL-12R expression but is also required to render JAKs/STAT4 responsive to IL-12 stimulation.

Effects of isoproterenol on spontaneous excitations in detrusor smooth muscle cells of the guinea pig.

PURPOSE: Because beta-adrenoceptor agonists would be a useful tool for the pharmacological treatment of unstable bladder, we investigated the cellular mechanisms underlying beta-adrenoceptor mediated inhibition on spontaneous excitation in detrusor smooth muscle. MATERIALS AND METHODS: Detrusor smooth muscle bundles were isolated from guinea pig bladders. Changes in membrane potential were recorded using an intracellular recording technique. In preparations loaded with the calcium indicator fura-PE3 changes in the concentration of intracellular calcium ions were measured simultaneously with membrane potential. Effects of isoproterenol on spontaneous changes in the membrane potential and intracellular Ca(2+) were examined RESULTS: Detrusor smooth muscle cells exhibited spontaneous action potentials that were associated with transient increases in intracellular Ca(2+) (calcium transients). Isoproterenol, which hyperpolarized the membrane, prevented action potentials and calcium transients. This induced inhibition of calcium transients was not affected by cyclopiazonic acid. Isoproterenol induced hyperpolarization was inhibited by inhibitors of protein kinase A, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride and Rp-adenosine-3',5'-cyclic phosphorothioate. Hyperpolarization was blocked by a solution containing 30 mM. potassium but not by a range of potassium channel blockers. Ouabain and a solution of 0.5 mM. potassium also inhibited hyperpolarization. CONCLUSIONS: Our results suggest that isoproterenol prevented spontaneous action potential discharges and associated calcium transients through the activation of protein kinase A. The isoproterenol induced inhibition of intracellular Ca(2+) largely depends on the prevention of spontaneous action potentials since the contribution of the intracellular calcium store was small. Isoproterenol hyperpolarizes the membrane, probably by stimulating sodium pump activity.

Properties of spontaneous electrical activity in smooth muscle of the guinea-pig renal pelvis.

In the guinea-pig renal pelvis, most smooth muscle cells examined (>90%), using a conventional microelectrode, had a resting membrane potential of about -50 mV and produced spontaneous action potentials with initial fast spikes and following plateau potentials. The remainder (<10%) had a resting membrane potential of about -40 mV and produced periodical depolarization with slow rising and falling phases. Experiments were carried out to investigate the properties of spontaneous action potentials. The potentials were abolished by nifedipine, suggesting a possible contribution of voltage-gated Ca(2+) channels to the generation of these potentials. Niflumic acid and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), inhibitors of Ca(2+)-activated Cl(-) channels, showed different effects on the spontaneous action potentials, and the former but not the latter inhibited the activities, raised the question of an involvement of Cl(-) channels in the generation of these activities. Depleting internal Ca(2+) stores directly with caffeine or indirectly by inhibiting Ca(2+)-ATPase at the internal membrane with cyclopiazonic acid (CPA) prevented the generation of spontaneous activity. Chelating intracellular Ca(2+) by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) increased the amplitude of the spike component of spontaneous activity. Indomethacin inhibited the spontaneous activity, whereas prostaglandin F(2 alpha) enhanced it. The results indicate that in smooth muscle of the renal pelvis, the generation of spontaneous activity is causally related to the activation of voltage-gated Ca(2+) channels through which the influx of Ca(2+) may trigger the release of Ca(2+) from the internal stores to activate a set of ion channels at the membrane. Endogenous prostaglandins may be involved in the initiation of spontaneous activity.

Pranidipine enhances relaxation produced by endothelium-derived relaxing factor in carotid artery.

The effects of pranidipine, a novel dihydropyridine-type Ca(2+)-channel antagonist, on acetylcholine-induced endothelium-dependent relaxation were investigated in isolated carotid artery of the guinea-pig. In arteries contracted with high-K(+) solution ([K(+)](0)=28.8 mM) containing noradrenaline, the relaxation was inhibited by N(omega)-nitro-L-arginine, indicating an involvement of endothelium-derived relaxing factor. Pranidipine (10(-9)-10(-7) M) augmented the relaxation in a concentration-dependent manner. Sodium nitroprusside produced a relaxation in arteries contracted with high-K(+) solution containing noradrenaline, in an endothelium-independent manner, and the relaxation was enhanced by pranidipine. 1H-[1,2,4] oxadiazolo [4, 3-a] quinoxalin-l-one (ODQ), an inhibitor of nitric oxide-sensitive guanylate cyclase, attenuated the relaxation produced by acetylcholine or sodium nitroprusside. In the presence of ODQ, pranidipine did not enhance the acetylcholine-induced relaxation. The relaxation produced by endothelium-derived hyperpolarizing factor was inhibited by pranidipine, with no alteration of the hyperpolarization. Thus, pranidipine augments the nitric oxide-induced relaxation, possibly by enhancing the mechanisms related to cyclic GMP.

Developmental stage-specific multi-subunit plastid RNA polymerases (PEP) in wheat.

Most photosystem I and II plastid genes are transcribed by a plastid encoded Escherichia coli-like RNA polymerase (PEP). In this study, we show that both promoter selectivity and light-dependency of PEP change dramatically during development in wheat leaves. In the leaf tip, psbA and psbD promoter activities are light induced, whilst psbC, psbE and 16S rRNA promoters do not function efficiently irrespective of light conditions. In contrast to the leaf tip, in the basal portion all PEP promoters studied function in the dark as well as the light, except for psbD. Using in vitro transcription, we found that PEP in the illuminated leaf tip can initiate transcription from the -35 destructed psbA promoter, but the -35 element is essential for transcription in the basal portion. There is an extended -10 element in the psbA promoter, recognized by the PEP in the illuminated leaf tip or purified sigma 70-type Escherichia coli RNA polymerase but not by the PEP in the leaf base. These results suggest that during wheat leaf development, PEP in the leaf base that is functional for most PEP promoters even in the dark is replaced by the light-dependent PEP selectively transcribing the psbA and psbD promoters.

Circadian-regulated expression of a nuclear-encoded plastid sigma factor gene (sigA) in wheat seedlings.

The activity of a light-responsive psbD promoter in plastids is known to be regulated by a circadian clock. However, the mechanism of the circadian regulation of the psbD light-responsive promotor, which is recognized by an Escherichia coli-type RNA polymerase, is not yet known. We examined the time course of mRNA accumulation of two E. coli-type RNA polymerase subunit genes, sigA and rpoA, under a continuous light condition after 12 h light/12 h dark entrainment. Accumulation of the sigA mRNA was found to be regulated by a circadian clock, while rpoA mRNA did not show any significant oscillation throughout the experiment.

Biometric confirmation of the Hirschberg ratio in strabismic children.

PURPOSE: In the Hirschberg eye position test, the ratio of strabismic angle to decentration of the corneal reflex is dependent on two biometric parameters of the eye: the radius of the corneal curvature and the depth of the anterior chamber. This study was designed to confirm whether the Hirschberg conversion ratio (HR) previously determined for adults can be used for children of various ages despite structural growth of the eye. METHODS: For 262 eyes of 131 children with strabismus (age range, 6 months to 11 years), the radius of the corneal curvature was measured with an auto-keratometer and the anterior chamber depth with an A-scan ultrasound unit under general anesthesia before the surgery. Using these measurements, the HR was computed on the basis of a geometric model. RESULTS: The calculated HR was constant across the age range, and the mean+/-SD was 19.9+/-1.9 prism diopters/mm (95% confidence interval, 16.1-23.6 prism diopters/mm). The ratios for the two eyes in each subject showed good correlation (R = 0.854, P = 0.0001). Neither of the biometric measurements was significantly correlated with age, although considerable scatter of the measurements was observed. CONCLUSIONS: These results indicate that the averaged HR can be applied in children regardless of the patient's age, although intersubject variance of the ratio should be taken into account.

Circadian-regulated transcription of the psbD light-responsive promoter in wheat chloroplasts.

The level of mRNAs derived from the plastid-encoded psbD light-responsive promoter (LRP) is controlled by a circadian clock(s) in wheat (Triticum aestivum). The circadian oscillations in the psbD LRP mRNA level persisted for at least three cycles in continuous light and for one cycle in continuous dark, with maxima in subjective morning and minima in subjective early night. In vitro transcription in chloroplast extracts revealed that the circadian cycles in the psbD LRP mRNA level were dominantly attributed to the circadian-regulated transcription of the psbD LRP. The effects of various mutations introduced into the promoter region on the psbD LRP activity in vitro suggest the existence of two positive elements located between -54 and -36, which generally enhance the transcription activity, and an anomalous core promoter structure lacking the functional "-35" element, which plays a crucial role in the circadian fluctuation and light dependency of psbD LRP transcription activity.

Blockade by 18beta-glycyrrhetinic acid of intercellular electrical coupling in guinea-pig arterioles.

1. Intercellular electrical communication between smooth muscle and endothelial cells was examined in guinea-pig mesenteric arterioles using the whole-cell patch-clamp method. The time course of the current required to impose a 10 mV voltage clamp step was used to determine the extent of electrical coupling between them. Currents recorded from both smooth muscle and endothelial cells relaxed in a multi-exponential manner, indicating the existence of electrical coupling between cells. 2. 18beta-Glycyrrhetinic acid, a gap junction blocker, quickly blocked electrical communication at 40 microM, while neither heptanol nor octanol did so at concentrations of up to 1 mM. 3. In the current clamp mode, repetitive spikes, induced by 10 mM Ba2+ solutions, could be recorded from both kinds of cells. After blocking gap junctions, spikes could only be recorded from the smooth muscle cell layer, indicating that they had been conducted through myoendothelial junctions. 4. In endothelial cells, acetylcholine (ACh, 3 microM) induced hyperpolarizing responses, which had two phases (an initial fast and a second slower phase) in the current clamp condition. This ACh response persisted in the presence of 18beta-glycyrrhetinic acid, although this compound seemed to make the membrane slightly leaky. 5. After blocking gap junctions, the membrane potential of a single cell in a multicellular preparation could be well clamped. Thus, 18beta-glycyrrhetinic acid may be useful in studying the function of both arteriolar smooth muscle and endothelial cells while they remain located within a multicellular preparation.

[Clinical efficacy of sairei-to in prevention of recurrence of urethral stenosis: report of two cases].

Sairei-to has been reported to inhibit granulation and fibroblast proliferation. We administered Sairei-to (9.0 g/day) to two 77-year-old men with repeated urethral stenosis after transurethral resection of prostate (TUR-P) and examined its clinical effects. Urethral stricture did not recur in these patients for 7 to 8 months. There were no side effects in these patients. Sairei-to is suggested to be useful to prevent recurrence of urethral stricture after a transurethral procedure.

Characterization of dynamics of the psbD light-induced transcription in mature wheat chloroplasts.

Dynamical aspects of three chloroplast promoters responding to change in light condition were examined in mature chloroplasts of wheat (Triticum aestivum) by in vitro transcription. The wheat psbD/C operon has four distinct promoters, two of which named as D/C-3 and D/C-4 promoters dominantly function in mature chloroplasts to produce the mRNAs encoding D2/CP43 and CP43 alone, respectively. Activity of the D/C-3 promoter in mature chloroplasts was reduced to less than 30% by 24 h dark adaptation and recovered by re-illumination to the original level within 30 to 60 min. The activation of the D/C-3 promoter which requires de novo cytoplasmic protein synthesis was induced by low fluence of light (e.g. 16 microE m(-2) s(-1)), but the extent of activation increased with increasing light fluence. The accumulation of mRNAs from the D/C-3 promoter saturated at 2- to 3-fold higher level within 2 h when the dark-adapted seedlings were transferred to the light at 72 microE m(-2) s(-1), concomitant with the increase in rate of D2 synthesis, suggesting that synthesis of D2 in mature chloroplasts is controlled via the D/C-3 promoter activity in a light-dependent way. Activity of the D/C-4 promoter slightly increased in the dark and decreased in the light. Effect of light on the psbA promoter activity was not observed at all in mature chloroplasts. In vitro transcriptional analysis of the D/C-3 promoter with 5' deletion mutations revealed that at least two cis elements which are located within the sequences of -78 to -47 and -46 to -29 of the transcription initiation site, respectively, act as enhancing elements in the D/C-3 promoter. The light-switching element of the transcription, however, was suggested to be located in the core promoter sequence downstream of the -35 element.

Metabolism of lidocaine by purified rat liver microsomal cytochrome P-450 isozymes.

The metabolism of lidocaine was studied using rat liver microsomes or a reconstituted lidocaine monooxygenase system with one of eight forms of cytochrome P-450 purified from liver microsomes from untreated- (P450 UT-2 and UT-5), phenobarbital- (P450 PB-1, PB-2, PB-4, and PB-5) or 3-methylcholanthrene- (P450 MC-1 and MC-5) treated rats. A reverse phase high-performance liquid chromatography system capable of simultaneously assaying four major lidocaine metabolites, namely, monoethylglycinexylidide (MEGX), 3-hydroxylidocaine (3-OH LID), methylhydroxylidocaine (Me-OH LID) and glycinexylidide (GX), was employed to determine the rate of formation of each metabolite. Untreated microsomes generated MEGX, Me-OH LID, and 3-OH LID, but the formation of GX was not detected. In male rat liver microsomes, MEGX was the major metabolite of lidocaine when a concentration of 1 mM was employed. The formation of MEGX and Me-OH LID was increased significantly (P less than 0.01) by microsomes from phenobarbital-treated rats, and the formation of 3-OH LID was increased with 3-methylcholanthrene. The study with the reconstituted system with purified cytochrome P-450 isozymes revealed that all eight forms of cytochrome P-450 used have an ability to N-deethylate lidocaine to form MEGX. Among these isozymes, cytochrome P450 PB-4 and P450 UT-2 showed a higher turnover number for the formation of MEGX. Me-OH LID was formed exclusively by P450 PB-5, and 3-OH LID exclusively by P450 MC-1. Selectivity of cytochrome P450 PB-5 for aromatic methyl hydroxylation of lidocaine was confirmed by an inhibition study; formation of Me-OH LID by microsomes of rats treated with phenobarbital was inhibited completely by antibody against P450 PB-5. It was concluded that different cytochrome P-450 isozymes metabolize lidocaine with a different rate and different position selectivities. Since a specific substrate of cytochrome P450 PB-5 (P-450e) is not known, lidocaine may be a useful substrate for the identification of P450 PB-5.