Polymerization of room-temperature ionic liquid monomers by electron beam irradiation with the aim of fabricating three-dimensional micropolymer/nanopolymer structures.

A novel method for fabricating microsized and nanosized polymer structures from a room-temperature ionic liquid (RTIL) on a Si substrate was developed by the patterned irradiation of an electron beam (EB). An extremely low vapor pressure of the RTIL, 1-allyl-3-ethylimidazolium bis((trifluoromethane)sulfonyl)amide, allows it to be introduced into the high-vacuum chamber of an electron beam apparatus to conduct a radiation-induced polymerization in the nanoregion. We prepared various three-dimensional (3D) micro/nanopolymer structures having high aspect ratios of up to 5 with a resolution of sub-100 nm. In addition, the effects of the irradiation dose and beam current on the physicochemical properties of the deposited polymers were investigated by recording the FT-IR spectra and Young's modulus. Interestingly, the overall shapes of the obtained structures were different from those prepared in our recent study using a focused ion beam (FIB) even if the samples were irradiated in a similar manner. This may be due to the different transmission between the two types of beams as discussed on the basis of the theoretical calculations of the quantum beam trajectories. Perceptions obtained in this study provide facile preparation procedures for the micro/nanostructures.

Tuning of the fluorescence wavelength of CdTe quantum dots with 2 nm resolution by size-selective photoetching.

Photoetching of CdTe nanocrystals was applied to thiol-capped CdTe quantum dots (QDs) to control their fluorescence wavelength. CdTe QDs with a high quantum yield (49%) were synthesized in aqueous solution, and they were successfully photoetched in strong alkaline (pH = 13.5) conditions. When monochromatic light was used, size-selective photoetching could be conducted; the photoetching proceeded until the band gap energy of the CdTe QDs increased to the energy corresponding to the wavelength of the irradiating light. As a result, a good linear relationship was obtained between the wavelength of the irradiating light and that of the fluorescence peak. The resulting CdTe QDs exhibited a fluorescence peak with an FWHM value as small as 23.5 nm, indicating preparation of highly monodispersed nanocrystals. The high quantum yield (ca. 45%) was maintained after the photoetching. Very fine tuning of the fluorescence wavelength with 2 nm resolution was achieved by changing the wavelength of the irradiating light by 2 nm. Theoretical calculation of the quantum size effects (effective mass approximation) predicts that a difference in the band gap fluorescence wavelength of 2 nm corresponds to a change in particle diameter of ca. 0.02 nm.

Soft-copy reading in digital mammography of mass: diagnostic performance of a 5-megapixel cathode ray tube monitor versus a 3-megapixel liquid crystal display monitor in a diagnostic setting.

BACKGROUND: Liquid crystal display (LCD) monitors and cathode ray tube (CRT) monitors are currently the two most common types used in digital mammography systems. The appropriate selection of a monitor is very important and requires balancing the monitor's performance and its cost. A previous study of soft-copy reading in digital mammography of microcalcifications showed that 3-megapixel (M) LCD monitors were similar in diagnostic performance to 5M CRT monitors in a diagnostic setting. PURPOSE: To compare 5M CRT monitors with 3M LCD monitors for soft-copy reading of digital mammography of a mass in a diagnostic setting. MATERIAL AND METHODS: Seventy mass lesions having undergone either breast biopsies or definitive surgery (46 malignant and 24 benign) and 30 normal cases were recruited into the study. The median size of the lesions was 16 mm (range 7-20 mm). The digital mammograms in the 100-case set were assigned to two blocks, block A (50 cases) and block B (50 cases), for 5M CRT and 3M LCD monitors. A single radiologist read all 100 cases with both types of monitors, starting with the images in block A on the 5M CRT monitors and then the images in block B on the 3M LCD monitors. The radiologist analyzed the soft-copy images on 5M CRT and 3M LCD monitors with 5 months between the interpretations to reduce the effects of learning and memory. Again, the reader started with the images in block A on the 3M LCD monitors and then read the images in block B on the 5M CRT monitors. A five-point rating scale for the probability of malignancy was used for interpreting the soft-copy mammograms. The mass descriptor was scored on a six-point scale. Breast density was scored on a four-point scale. The positive predictive value (PPV) and negative predictive value (NPV) were calculated based on the criteria of the Breast Imaging Reporting and Data System. The interpretation time was also measured. RESULTS: No significant difference was observed in the probability of malignancy (P=1), mass descriptor (P=0.317), and breast density (P=0.739). The PPV and NPV of soft-copy reading on the 5M CRT monitors were 91% (42/46) and 94% (51/54), respectively, identical to the results using 3M LCD monitors. The total interpretation time averaged 62 s for the 5M CRT monitors and 60 s for the 3M LCD monitors (P<0.0001). CONCLUSION: Soft-copy reading of a digital mammography of mass with 3M LCD monitors was similar in diagnostic performance to 5M CRT monitors in this study. On the basis of the results of this and a previous study, 3M LCD monitors can replace 5M CRT monitors without any loss in the ability to diagnose digital mammograms.

Inorganic polyphosphate: a possible stimulant of bone formation.

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

Basic fibroblast growth factor induces interleukin-6 synthesis in osteoblasts: autoregulation by protein kinase C.

We previously reported that basic fibroblast growth factor (bFGF) stimulates both phospholipases C and D via independent pathways in osteoblastlike MC3T3-E1 cells. In this study, we investigated the effect of bFGF on interleukin-6 (IL-6) synthesis in these cells. bFGF stimulated the IL-6 synthesis dose-dependently in the range between 1 and 30 ng/ml. The depletion of extracellular Ca2+ by EGTA suppressed the bFGF-induced IL-6 synthesis. TMB-8, an inhibitor of intracellular Ca2+ mobilization, also inhibited the IL-6 synthesis by bFGF. bFGF stimulated the Ca2+ influx from extracellular space. Genistein, a tyrosine kinase inhibitor, suppressed the bFGF-induced Ca2+ influx. Staurosporine, an inhibitor for protein kinases, enhanced the bFGF-induced IL-6 synthesis. Calphostin C, a highly potent and specific inhibitor for protein kinase C (PKC), also enhanced the IL-6 synthesis by bFGF. The bFGF-induced IL-6 synthesis was amplified in PKC down-regulated cells. U-73122, a phospholipase C inhibitor, enhanced the bFGF-induced IL-6 synthesis. Propranolol, a phosphatidic acid phosphohydrolase inhibitor, also enhanced the IL-6 synthesis by bFGF. These results strongly suggest that bFGF stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilization in osteoblastlike cells, and that the IL-6 synthesis by bFGF is autoregulated due to PKC activation.

p38 MAP kinase is involved in the signalling of sphingosine in osteoblasts: sphingosine inhibits prostaglandin F(2alpha)-induced phosphoinositide hydrolysis.

We previously showed that sphingosine inhibits prostaglandin F(2alpha) (PGF(2alpha))-stimulated interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of sphingosine on phospholipase C-catalyzing phosphoinositide hydrolysis induced by PGF(2alpha) in these cells. Sphingosine inhibited the inositol phosphates formation by PGF(2alpha) or NaF, a GTP-binding protein activator. Sphingosine induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase but did not affect the phosphorylation of p42/p44 MAP kinase. SB203580 and PD169316, inhibitors of p38 MAP kinase, rescued the inhibitory effect of sphingosine on the formation of inositol phosphates by PGF(2alpha) or NaF. These results indicate that sphingosine inhibits PGF(2alpha)-induced phosphoinositide hydrolysis by phospholipase C via p38 MAP kinase in osteoblasts.

p42/p44 mitogen-activated protein kinase activation is involved in prostaglandin F2alpha-induced interleukin-6 synthesis in osteoblasts.

Prostaglandin F2alpha (PGF2alpha) significantly induced p42/p44 mitogen-activated protein (MAP) kinase activity in osteoblast-like MC3T3-E1 cells. PD98059, a selective inhibitor of MAP kinase kinase, inhibited PGF2alpha-induced interleukin-6 (IL-6) synthesis as well as PGF2alpha-induced p42/p44 MAP kinase activation. PD98059 suppressed the IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, or NaF, an activator of heterotrimeric GTP-binding protein, as well as the p42/p44 MAP kinase activation by TPA or NaF. Calphostin C, a highly potent and specific inhibitor of PKC, inhibited the PGF2alpha-induced p42/p44 MAP kinase activity. These results strongly suggest that PKC-dependent p42/p44 MAP kinase activatioin is involved in PGF2alpha-induced IL-6 synthesis in osteoblasts.

Effect of ceramide on interleukin-6 synthesis in osteoblast-like cells.

We previously showed that prostaglandin (PG) E1 stimulates the synthesis of interleukin-6 (IL-6) through activation of protein kinase (PK) A in osteoblast-like MC3T3-E1 cells and that PGF2alpha induces IL-6 synthesis through PKC activation. In other studies, we demonstrated that thrombin stimulates IL-6 synthesis, which depends on intracellular Ca2+ mobilisation in these cells and that tumour necrosis factor-alpha (TNF) induces IL-6 synthesis through sphingosine 1-phosphate, a product of sphingomyelin turnover. In the present study, among sphingomyelin metabolites, we examined the effect of ceramide on the IL-6 synthesis induced by various agonists in MC3T3-E1 cells. C2-ceramide, a cell-permeable ceramide analogue, suppressed the PGE1-induced IL-6 synthesis. C2-ceramide inhibited the IL-6 synthesis induced by PGF2alpha or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. C2-ceramide reduced the IL-6 synthesis induced by cholera toxin, forskolin or dibutyryl cAMP. C2-ceramide inhibited the IL-6 synthesis induced by thrombin. The IL-6 synthesis stimulated by thapsigargin, which is known to stimulate Ca2+ mobilisation from intracellular Ca2+ stores, or A23187, a Ca-ionophore, was also inhibited by C2-ceramide. C2-ceramide did not affect the IL-6 synthesis induced by interleukin-1. On the contrary, C2-ceramide enhanced the TNF-induced IL-6 synthesis. D,L-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, inhibited the enhancement by C2-ceramide as well as the TNF-effect. These results strongly suggest that ceramide modulates the IL-6 synthesis stimulated by various agonists in osteoblasts.

Endothelin-1 induces vascular endothelial growth factor synthesis in osteoblasts: involvement of p38 mitogen-activated protein kinase.

We previously reported that endothelin-1 (ET-1) stimulates p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of ET-1 on the synthesis of vascular endothelial growth factor (VEGF) in these cells. ET-1 significantly stimulated VEGF secretion time-dependently 18 hours after the stimulation. The stimulatory effect was dose-dependent in the range between 0.1 nM and 0.1 micro;M. BQ123, an antagonist of endothelin(A) (ET(A)) receptor, inhibited the ET-1-induced VEGF secretion. The ET-1-induced VEGF secretion was suppressed by SB203580 and PD169316, inhibitors of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p44/p42 MAP kinase. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC)-activating phorbol ester, stimulated VEGF secretion. Calphostin C, a specific PKC inhibitor, suppressed the VEGF secretion by ET-1. TPA-induced VEGF secretion was suppressed by SB203580. Taken together, our results strongly suggest that ET-1 stimulates VEGF synthesis via ET(A) receptor in osteoblasts and that p38 MAP kinase is involved at a point downstream from PKC in the VEGF synthesis.

Involvement of protein kinase C activation in endothelin-1-induced secretion of interleukin-6 in osteoblast-like cells.

We previously reported that endothelin-1 (ET)-1 stimulates phospholipase D (PLD) independently of phosphoinositide hydrolysis in osteoblast-like MC3T3-E1 cells. In the present study, we examined the effect of ET-1 on the secretion of interleukin-6 (IL-6) and the involvement of protein kinase C (PKC) activation in the IL-6 secretion in these cells. ET-1 significantly stimulated IL-6 secretion time-dependently up to 72 h. The stimulative effect was dose-dependent in the range between 1 nM and 1 microM. BQ123, a selective antagonist of endothelinA (ETA) receptor, inhibited the ET-1-induced IL-6 secretion. On the contrary, BQ788, a selective antagonist of endothelinB (ETB) receptor, had no effect. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC-activating phorbol ester, significantly stimulated IL-6 secretion. However, 4 alpha-phorbol 12,13-didecanoate, a PKC-nonactivating phorbol ester, did not affect IL-6 secretion. The effect of a combination of ET-1 and TPA on IL-6 secretion was not additive. Calphostin C, a specific PKC inhibitor, significantly inhibited the ET-1-induced IL-6 secretion. Both ET-1- and TPA-induced IL-6 secretion were reduced in PKC downregulated MC3T3-E1 cells. These results strongly suggest that ET-1 stimulates IL-6 secretion via ETA receptor in osteoblast-like cells and that PKC activation is involved in the ET-1-induced IL-6 secretion.

Mechanism of prostaglandin D(2)-stimulated heat shock protein 27 induction in osteoblasts.

We previously showed that prostaglandin D(2) (PGD(2)) stimulates activation of protein kinase C (PKC). We investigated whether PGD(2) stimulates the induction of heat shock protein (HSP) 27 and HSP70 in osteoblast-like MC3T3-E1 cells and the mechanism underlying the induction. PGD(2) increased the levels of HSP27 while having little effect on HSP70 levels. PGD(2) stimulated the accumulation of HSP27 dose dependently in the range between 10 nM and 10 microM. PGD(2) induced an increase in the levels of mRNA for HSP27. The PGD(2)-stimulated accumulation of HSP27 was reduced by staurosporine or calphostin C, inhibitors of PKC. PGD(2) induced the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase. The HSP27 accumulation induced by PGD(2) was significantly suppressed by PD98059, an inhibitor of the upstream kinase of p44/p42 MAP kinase, or SB203580, an inhibitor of p38 MAP kinase. Calphostin C suppressed the PGD(2)-induced phosphorylation of p44/p42 MAP kinase and p38 MAP kinase. PD98059 or SB203580 suppressed the PGD(2)-increased levels of mRNA for HSP27. These results strongly suggest that PGD(2) stimulates HSP27 induction through p44/p42 MAP kinase activation and p38 MAP kinase activation in osteoblasts and that PKC acts at a point upstream from both the MAP kinases.

Deletion of serum lectin-reactive alpha-fetoprotein by acyclic retinoid: a potent biomarker in the chemoprevention of second primary hepatoma.

A goal of cancer chemoprevention is the deletion of latent premalignant or malignant clones before they expand to a clinically detectable tumor. However, such clonal deletion has not been demonstrated in clinical studies. We have evaluated serum levels of lectin-reactive alpha-fetoprotein (AFP-L3), which suggests the presence of latent hepatoma cells, in a randomized controlled trial that used acyclic retinoid to prevent second primary hepatomas in patients who had received treatments that cured initial hepatomas. The trial involved 21 patients in each acyclic retinoid (600 mg daily) and placebo group and consisted of a 12-month period of drug administration and a subsequent follow-up period. Serum AFP-L3 was determined at entry and at the end of the 12-month treatment period using lectin-affinity electrophoresis and antibody-affinity blotting. Although neither treatment affected serum levels of total AFP, acyclic retinoid significantly reduced AFP-L3 levels after a 12-month administration (P < 0.01). Acyclic retinoid not only deleted AFP-L3 from patients who had been positive for AFP-L3 at entry but also prevented the appearance of AFP-L3 in patients who had been negative at entry (P < 0.01). In contrast, placebo significantly raised the incidence of AFP-L3-positive patients after a 12-month administration from that at entry (P < 0.05). Patients positive for AFP-L3 after a 12-month treatment had a significantly higher risk of second primary hepatomas in the subsequent follow-up period (P = 0.03). Acyclic retinoid may have deleted a clone of latent hepatoma cells producing AFP-L3 and thereby inhibited second primary hepatomas. Serum AFP-L3 may be a useful intermediate biomarker in the chemoprevention of second primary hepatomas by acyclic retinoid.

Basic fibroblast growth factor stimulates vascular endothelial growth factor release in osteoblasts: divergent regulation by p42/p44 mitogen-activated protein kinase and p38 mitogen-activated protein kinase.

We previously showed that basic fibroblast growth factor (bFGF) activates p38 mitogen-activated protein (MAP) kinase via Ca2+ mobilization, resulting in interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of bFGF on the release of vascular endothelial growth factor (VEGF) in these cells. bFGF stimulated VEGF release dose dependently in the range between 10 and 100 ng/ml. SB203580, an inhibitor of p38 MAP kinase, markedly enhanced the bFGF-induced VEGF release. bFGF induced the phosphorylation of both p42/p44 MAP kinase and p38 MAP kinase. PD98059, an inhibitor of upstream kinase of p42/p44 MAP kinase, reduced the VEGF release. SB203580 enhanced the phosphorylation of p42/p44 MAP kinase induced by bFGF. The enhancement by SB203580 of the bFGF-stimulated VEGF release was suppressed by PD98059. The depletion of extracellular Ca2+ by [ethylenebis(oxyethylenenitrilo)]tetracetic acid (EGTA) or 1,2-bis-(O-aminophinoxy)-ethane-N,N,N,N-tetracetic acid tetracetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca2+, suppressed the bFGF-induced VEGF release. A23187, a Ca ionophore, or thapsigargin, known to induce Ca2+ release from intracellular Ca2+ store, stimulated the release of VEGF by itself. A23187 induced the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase. PD98059 suppressed the VEGF release induced by A23187. SB203580 had little effect on either A23187-induced VEGF release or the phosphorylation of p42/p44 MAP kinase by A23187. These results strongly suggest that bFGF stimulates VEGF release through p42/p44 MAP kinase in osteoblasts and that the VEGF release is negatively regulated by bFGF-activated p38 MAP kinase.

Sphingosine 1-phosphate induces heat shock protein 27 via p38 mitogen-activated protein kinase activation in osteoblasts.

We previously showed that sphingosine 1-phosphate acts as a second messenger for tumor necrosis factor alpha-induced interleukin-6 synthesis in osteoblast-like MC3T3-E1 cells and that the synthesis by sphingosine 1-phosphate is dependent on p42/p44 mitogen-activated protein (MAP) kinase activation. In the present study, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27) in MC3T3-E1 cells. Not C2-ceramide, but sphingosine and sphingosine 1-phosphate significantly induced HSP27 accumulation dose dependently in the range between 1microM and 30 microM. DL-threo-dihydrosphingosine, an inhibitor of sphingosine kinase, markedly inhibited the sphingosine-induced HSP27 accumulation. Sphingosine 1-phosphate induced increase in the levels of the mRNA for HSP27. Sphingosine 1-phosphate stimulated the phosphorylation of p38 MAP kinase. The sphingosine 1-phosphate-induced HSP27 accumulation was dose dependently suppressed by SB203580, an inhibitor of p38 MAP kinase, but not PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase. SB203580 reduced the sphingosine 1-phosphate-induced increase of mRNA for HSP27. These results strongly suggest that sphingosine 1-phosphate-stimulated HSP27 induction is mediated via p38 MAP kinase activation in osteoblasts.

Mitogen-activated protein (MAP) kinases are involved in interleukin-1 (IL-1)-induced IL-6 synthesis in osteoblasts: modulation not of p38 MAP kinase, but of p42/p44 MAP kinase by IL-1-activated protein kinase C.

We previously reported that interleukin-1alpha (IL-1alpha)-induced activation of protein kinase C (PKC) via phosphatidylcholine-specific phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1alpha itself in osteoblast-like MC3T3-E1 cells. In the present study, we further investigated the mechanism behind IL-1alpha-induced IL-6 synthesis in MC3T3-E1 cells. IL-1alpha time-dependently stimulated the phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase. PD98059, a specific inhibitor of the upstream kinase that activates p42/p44 MAP kinase, inhibited the IL-1alpha-induced IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase induced by IL-1alpha. SB203580, a specific inhibitor of p38 MAP kinase, also reduced both the phosphorylation of p38 MAP kinase and the IL-6 synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed the IL-1alpha-induced IL-6 synthesis. Calphostin C, a specific inhibitor of PKC, or D-609, a specific inhibitor of PC-PLC, significantly enhanced the IL-1alpha-induced phosphorylation of p42/p44 MAP kinase without affecting the phosphorylation of p38 MAP kinase. The phosphorylation of p42/p44 MAP kinase by IL-1alpha was markedly increased in PKC-down-regulated MC3T3-E1 cells. Neither 12-O-tetradecanoylphorbol-13-acetate, known to be an activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the phosphorylation of p38 MAP kinase induced by IL-1alpha. These results strongly suggest that IL-1alpha-induced IL-6 synthesis is mediated via activations of both p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC activated by IL-1alpha itself negatively regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.

Triiodothyronine modulates interleukin-6 synthesis in osteoblasts: inhibitions in protein kinase A and C pathways.

In osteoblast-like MC3T3-E1 cells, we recently reported that PGE1 and PGF2alpha induce interleukin (IL)-6 synthesis via activation of protein kinase A and protein kinase C, respectively. Moreover, in the case of IL-1-induced IL-6 synthesis in these cells, we showed that protein kinase C activation by IL-1 limits the IL-6 synthesis. In the present study, we investigated the effect of T3 on IL-6 synthesis induced by these agonists in MC3T3-E1 cells. T3, which by itself had little effect on IL-6 synthesis, significantly reduced the IL-6 synthesis induced by PGE1 in a dose-dependent manner in the range between 10 pM and 10 nM. T3 also reduced PGE1-induced activation of protein kinase A. T3 inhibited the IL-6 synthesis induced by cholera toxin, an activator of Gs, or forskolin, which directly activates adenylate cyclase. However, T3 did not affect (Bu)2cAMP-induced IL-6 synthesis. In addition, T3 reduced PGF2alpha-induced IL-6 synthesis dose dependently in the range between 10 pM and 10 nM. T3 also inhibited IL-6 synthesis induced by 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C. On the other hand, T3 markedly enhanced IL-1-induced IL-6 synthesis. This enhancement by T3 was potentiated in protein kinase C down-regulated cells. T3 hardly affected the protein kinase C activation induced by PGF2alpha or IL-1. These results strongly suggest that T3 modulates IL-6 synthesis at two points in osteoblasts as follows; one is exerted at the point between adenylate cyclase and protein kinase A, and the other is at a point downstream from protein kinase C activation.

Protective effect of apoptosis-inhibitory agent, N-tosyl-L-phenylalanyl chloromethyl ketone against ischemia-induced hippocampal neuronal damage.

Delayed neuronal death in the gerbil hippocampal CA1 sector occurs 48 to 72 hours after severe forebrain ischemia. DNA fragmentation is observed in the hippocampal CA1 neurons at around that time. We show here that an inhibitor of proteolytic process of apoptosis, N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), protected hippocampal neuronal damage by inhibition of the DNA fragmentation in a dose-dependent manner and that TPCK induced an apoptosis-regulating molecule, Bcl-2 protein, in the surviving neurons. These results suggest the prevention of apoptosis-related DNA fragmentation by TPCK may be an attractive therapeutic strategy for preserving hippocampal neurons from ischemic insult.

Kinetics and electrocardiographic changes after oral 3-OH-quinidine in healthy subjects.

3-OH-quinidine, a major quinidine metabolite, has been reported to have antiarrhythmic activity in animals and is suspected to contribute to the effect of quinidine in man. Four healthy subjects received 3-OH-quinidine in increasing oral doses (35, 100, 300 mg) to achieve serum concentrations in the range of those after quinidine dosing in patients. Blood and urine were collected up to 48 hours and blood pressure, heart rate, and averaged ECG complexes were recorded during 12 hours after dosing. Kinetic analysis revealed differences from published data for the parent drug. Renal clearance was 16 L/hr. The elimination t1/2 was 12.4 hours, substantially longer than that of quinidine. No systematic ECG changes were observed in two subjects with maximum concentrations of 55 and 215 micrograms/L. In the other two subjects who achieved higher maximum concentrations (447 and 918 micrograms/L), there was a significant relationship between the length of the corrected QT interval and the serum concentration of 3-OH-quinidine. These first dynamic results indicate that 3-OH-quinidine exerts effects in man resembling those of quinidine and may contribute to the antiarrhythmic activity of quinidine.

Kinetics and dynamics of quinidine-N-oxide in healthy subjects.

The pharmacokinetics of a major metabolite of quinidine in humans, quinidine-N-oxide, were investigated after single oral doses (3 to 15 mg) in four healthy subjects. The concentration in serum and urine was determined by an HPLC assay. Because of a small volume of distribution, the elimination half-life of quinidine-N-oxide was only 2.5 +/- 0.28 hours (mean +/- SD), considerably shorter than that of quinidine. The renal clearance was 1.3 +/- 0.3 L/hr. Only 13.9% +/- 3.7% of the dose was recovered in urine as unchanged compound for up to 12 hours. Two unidentified compounds with the same retention time as quinidine and 3-hydroxyquinidine were found in the urine samples of two subjects. The free fraction in serum was 3.3% +/- 0.83%. No systematic changes in heart rate-corrected QT interval were observed up to concentrations of 500 ng/ml. The results indicate that quinidine-N-oxide, in contrast to 3-hydroxyquinidine, does not possess quinidine-like pharmacologic activity.

Expression of multiple alpha1-antitrypsin-like genes in hibernating species of the squirrel family.

In the chipmunk, a mammalian hibernator, a 140 kDa protein complex found in the blood, drastically decreases in concentration during hibernation. This complex contains four species of proteins, HP-20, -25, -27 and -55. In the present study, cDNA clones coding for the chipmunk HP-55 were isolated from a liver cDNA library. Sequence analysis revealed that HP-55 is produced as a precursor protein of 413 amino acids (aa), that it has a signal peptide of 24 aa, and that it contains four potential N-glycosylation sites. The deduced aa sequence shows 63% identity with that of rat alpha1-antitrypsin (alpha1-AT); however, the sequence corresponding to the reactive center P1-P1' residues was found to be Met-Leu, whereas it is Met-Ser in the rat alpha1-AT. During screening of the chipmunk liver cDNA library, four other related classes of cDNA clones were obtained, each also coding for an alpha1-AT-like protein. In spite of more than 86% overall aa sequence identity among the five chipmunk alpha1-AT-like proteins, they are highly divergent in the putative reactive center region; the putative P1-P1' sequences are Met-Leu (HP-55 or CM55-ML), Met-Met (CM55-MM), Met-Ser (CM55-MS), Ser-Ile (CM55-SI) and Ser-Thr (CM55-ST). Each of the alpha1-AT-like protein mRNAs was expressed in chipmunk liver, and the HP-55 mRNA level was greatly reduced during hibernation. Genomic Southern blot analysis and screening of a liver cDNA library from another hibernating squirrel species, the ground squirrel, also revealed expression of multiple members of the alpha1-AT gene family, whereas analysis of a cDNA library from a non-hibernating species, the tree squirrel, found only a single alpha1-AT gene.