The temperature-dependent photoluminescence features of polycarbonate thin films doped with blue-phosphorescent molecules, either bis[(4,6-difluorophenyl)-pyridinato-N,C-2'] (picolinate) iridium (Flrpic) or bis(2-phenylpyridinato-N,C-2') (acetylacetonate) rhodium [(ppy)(2)Rh(acac)], which have an equivalent triplet energy of 2.64 eV, have been studied. The photoluminescence intensity of the Flrpic-doped polycarbonate thin film did not show any dependence on temperature. On the other hand, as for the (ppy)(2)Rh(acac)-doped polycarbonate thin film, decreasing photoluminescence intensity with increasing temperature (especially above 100K) was clearly visible. These results reflect that the internal heavy-atom effect of (ppy)(2)Rh(acac) is weaker than that of Flrpic. Furthermore, the steady-state and time-resolved photoluminescence spectra of tris(8-hydroxyquinoline) aluminum (Alq(3)) thin films heavily doped with Flrpic or (ppy)(2)Rh(acac) (50 wt.%) at 8K was studied. It was found that the enhanced phosphorescence from Alq(3) is mainly due not to the external heavy-atom effect by doping with the phosphorescent molecule but to the exothermic triplet energy transfer from the phosphorescent molecule to Alq(3).

Gonadal differentiation in amphibians is sensitive to steroids. The phenotypic sex can be changed by hormonal treatments, but the molecular mechanism for gonadal differentiation is not known. Up to date, many genes involved in gonadal differentiation have been isolated in vertebrates. Dmrt1, a gene that contains the DM-domain (Doublesex/Mab-3 DNA-binding motif), is considered to be one of the essential genes involved in the testicular differentiation cascade in mammals, birds, reptiles, and fish. However, this gene has not been isolated in amphibians yet. To elucidate its role(s) for gonadal differentiation in vertebrates, a molecular cloning of Dmrt1 in amphibians is urgent. In this study, we have successfully isolated a Dmrt1 homolog from the frog Rana rugosa testes cDNA library and examined its expression during gonadal differentiation and in sex-reversed gonads. The Dmrt1 mRNA was exclusively detected in testis among adult tissues by the RT-PCR analysis. The Dmrt1 was first expressed in the differentiating testis at stage XXV in which spermatogonia are only germ cells, and became stronger at later stages. Moreover, the Dmrt1 transcript was not detected during ovarian differentiation. However, this gene was clearly expressed in XX sex-reversed gonads caused by injection of testosterone into all-female tadpoles that have well-differentiated ovaries. Taken together, the results suggest that Dmrt1 is closely implicated in testicular, but not ovarian differentiation in amphibians.

Background: Localized aggressive periodontitis (LAgP) is associated with neutrophil dysfunction including defective chemotaxis and reduced calcium influx factor activity. Nitric oxide (NO) and its enzyme, nitric oxide synthase (NOS), have been suggested to be involved in chemotaxis. Some reports, however, were unable to detect either NO or NOS in human neutrophils. In this study, we focused on NOS activity in LAgP neutrophils and examined the involvement of NOS in chemotaxis of normal neutrophils and NOS activity in neutrophils from normal subjects and patients with LAgP.Methods: Neutrophils from 10 normal subjects and 10 LAgP patients were isolated from peripheral venous blood. Membrane associated-NOS (MA-NOS) and soluble NOS (S-NOS) were extracted from cells with or without FMLP stimulation. NOS activity was measured using the radiolabeled L-arginine to L-citrulline conversion assay.Results: N-omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly inhibited FMLP-induced chemotaxis (P<0.01) and dibutyryl cGMP, an activator of cGMP-dependent protein kinase, significantly attenuated the inhibition by L-NAME (P<0.01). Unstimulated and FMLP-stimulated MA-NOS activity in LAgP neutrophils was statistically significantly higher than that in normal neutrophils (P <0.05). S-NOS activity in LAgP neutrophils was higher than that in normal neutrophils.Conclusions: This study suggests that NOS is present in human neutrophils and may be involved in FMLP-induced chemotaxis in normal neutrophils. NOS activity is increased in LAgP and is negatively correlated to chemotaxis response.

BACKGROUND:Localized juvenile periodontitis (LJP) is an early-onset periodontal disease associated with neutrophil dysfunction, including defective chemotaxis, reduced protein kinase C (PKC) activity, and reduced calcium entry. These observations are important because reduced availability of cytosolic-free calcium concentration in the cell will have detrimental consequences for the numerous cytosolic calcium concentration-dependent pathways. In particular, there is a direct relationship between Ca2+ flux and the cell activation enzyme PKC. In this report, we focused on the mechanism of calcium entry, investigating a newly described molecule, calcium influx factor (CIF). CIF is thought to be a second messenger for the opening of membrane calcium channels when intracellular calcium stores are depleted. We examined CIF activity in neutrophils from normal subjects and LJP patients.METHODS:Neutrophils from 11 LJP patients, 3 adult periodontitis (AP) patients, and 12 normal subjects were isolated from peripheral venous blood. CIF was extracted with thapsigargin, a Ca2+-ATPase inhibitor, from isolated neutrophils and CIF activity measured using a 45CaCl2 uptake assay.RESULTS:The CIF activity in neutrophils from LJP patients ranged from 98.9 to 281.5 units/mg protein (mean = 180.2 +/- 56.3) and from 291.9 to 755.5 units/mg protein (mean = 528.8 +/- 153.8) in non-periodontal disease controls. CIF activity in AP patients was also measured and found to be similar to controls. The CIF activity in LJP patients was statistically significantly reduced compared to that in normal subjects (P <0.001).CONCLUSIONS:This study suggests that CIF activity may be an important determinant in neutrophil abnormalities in LJP.

12(R)-hydroxyeicosatetraenoic acid (HETE) shows biphasic increase in cytosolic free calcium concentration ([Ca2+]i) in rabbit and human neutrophils; the initial transient phase and the continuous falling phase. 12(S)-HETE was less potent in both species. BN50739, a platelet-activating factor (PAF) receptor antagonist, inhibited both phases of 12(R)-HETE-induced [Ca2+]i rise but did not affect leukotriene B4 (LTB4)-induced [Ca2+]i rise. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), a PAF synthesis inhibitor, and manoalide, a phospholipase A2 inhibitor, reduced 12(R)-HETE-induced [Ca2+]i rise. These blockers inhibited the continuous phase of [Ca2+]i rise induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP) with little effect on the initial phase. It had no significant effect on LTB4-induced [Ca2+]i rise. SC-41930, a LTB4-receptor antagonist, did not block 12-HETE-induced [Ca2+]i rise. In 12(R)-HETE-, FMLP- and LTB4-stimulated cells, accumulations of cell-associated PAF and released PAF were detected but not in unstimulated cells. BN50739 did not affect the accumulation of cell-associated PAF and release of PAF in 12(R)-HETE-stimulated cells. These results suggest that 12(R)-HETE-induced and partially, FMLP-induced, but not LTB4-induced [Ca2+]i rise are mediated by PAF, which is produced and released by stimulation of the cells by 12(R)-HETE and FMLP, respectively.

The soluble fractions of infected root canal contents (IRCC) were collected from about 300 human extracted teeth and examined for the presence of mononuclear cell (MNC) chemotaxis and cellular immunocompetence. IRCC showed remarkable chemotactic activity for polymorphonuclear leukocytes but a weak activity for MNC. However, generation of intrinsic MNC chemotaxis and induction of cellular immunity were confirmed in rats given repeated injections of IRCC.

Extracellular application of acid extract from platelet-activating factor- or thapsigargin-treated rabbit neutrophils induced a rise of cytosolic free calcium concentration ([Ca2+]i) in neutrophils and adrenal chromaffin cells suspended in Ca(2+)-containing, but not in Ca(2+)-deficient, medium. The ability of the extract to selectively induce Ca2+ entry was also confirmed by the increase in 45Ca2+ uptake and failure to stimulate Ca2+ release in digitonin-permeabilized neutrophils. 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited the extract-induced [Ca2+]i rise in a staurosporine (ST)-sensitive fashion, neither of which had any effect on its production. SK&F 96365 and econazole also reduced extract-induced Ca2+ entry. These results suggest that a Ca2+ entry-inducible substrate (calcium influx factor) is extracted from Ca2+ store-depleted neutrophils, and that its action may be regulated by protein kinase C and certain pharmacological agents.

12-O-Tetradecanoylphorbol-13-acetate (TPA) time-dependently inhibited the platelet-activating factor (PAF)-induced rise in cytosolic free calcium concentration ([Ca2+]i) in rabbit neutrophils, whereas staurosporine significantly enhanced it. Inositol 1,4,5-trisphosphate (IP3) induced Ca2+ release in digitonin-permeabilized cells but not in PAF-pretreated permeabilized cells. IP3-induced Ca2+ release was not affected by protein kinase C activators or inhibitors. In the cells pretreated with PAF and thapsigargin in Ca2+-deficient medium, stimulated Ca2+ entry was evoked by the subsequent addition of CaCl2. TPA inhibited the Ca2+ entry induced by PAF and thapsigargin in a staurosporine-reversible manner but not thapsigargin-induced [Ca2+]i elevation. These results suggest that protein kinase C negatively regulates PAF- and thapsigargin-induced rise in [Ca2 +]i possibly by inhibiting Ca2+ store depletion-induced Ca2+ entry.

12-Hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) has been reported to be a chemoattractant for human neutrophils. To assess its cellular mechanism, we focused on the effect of 12-HETE on cytosolic Ca2+ ([Ca2+](i)) and characterized the effect of 12-HETE on [Ca2+](i) in human neutrophils. 12(S)- and 12(R)-HETE increased [Ca2+](i) in the presence and absence of extracellular Ca2+ in a concentration-related fashion. The elevation of [Ca2+](i) by 12(R)-HETE was completely abolished by pertussis toxin treatment. U-73122, a selective phospholipase C inhibitor, depressed the 12(S)- and 12(R)-HETE-induced rise in [Ca2+](i) in the presence and absence of extracellular Ca2+. 12(R)-HETE resulted in the rapid production of inositol 1,4,5-trisphosphate (IP3). Furthermore, 12(R)-HETE elicited slight depolarization of neutrophils as assessed using the fluorescent dye bis-oxonol. These results provide evidence demonstrating the signal transduction pathway in human neutrophils after stimulation with 12-HETE and suggest that 12-HETE causes a rapid rise of [Ca2+](i) by mobilizing Ca2+ from an IP3-sensitive intracellular Ca2+ pool in human neutrophils.