Preservation of Adrenoceptor and Endothelin Receptor Mediated Vasoconstriction and of Endothelium-Dependent Relaxation after Cold Storage of Explanted Blood Vessels for ex vivo Analyses.

INTRODUCTION: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. METHODS: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. RESULTS: Storage in HEPES-supplemented Dulbecco's modified Eagle's medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E-8 vs. 7.5E-8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. CONCLUSION: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

Interactions between copper (II) and ß-amyloid peptide using capillary electrophoresis-ICP-MS: Kd measurements at the nanogram scale.

Although the interaction between the ß-amyloid peptide and copper (II) appears to play an important role in Alzheimer's disease, the affinity constant is still controversial and values are ranging from 107 to 1011 M-1. With the aim of clarifying this point, a complementary method, based on the capillary electrophoresis-ICP-MS hyphenation, was developed and competitive binding experiments were conducted in the presence of nitrilotriacetic acid. The effect of the capillary surface (neutral or positively charged) and nature of the buffer (Tris or Hepes) have been studied. Tris buffer was found to be inappropriate for such determination as it enhances the dissociation of copper (II) complexes, already occurring in the presence of an electric field in capillary electrophoresis. Using Hepes, a value of 1010 M-1 was found for the affinity of the small ß-amyloid peptide 1-16 for copper (II), which is in agreement with the values obtained for other proteins involved in neurodegenerative diseases. These constants were also determined in conditions closer to those of biological media (higher ionic strength, presence of carbonates).

A novel OFF-ON-OFF fluorescence probe based on coumarin for Al3+ and F- detection and bioimaging in living cells.

A novel fluorescence probe L2 based on coumarin has been designed and synthesized. The probe L2 can be used for relay recognition of metal ions Al3+ and anion F- in the aqueous HEPES buffer (0.05 M, pH = 7.4), and build a OFF-ON-OFF detection system. The probe showed high selectivity and sensitivity to target ions in the process of relay recognition, and the corresponding detection limit could be as low as 0.014 µM (Al3+) and 0.03 µM (F-). Besides, the geometry optimizations of probe L2 and [L2 + Al3+] complex were carried out using the Gaussian 16 program based on DFT, and the identification mechanism of the probe was also discussed by the mass spectrometry and theoretical calculations. Moreover, the probe has also been successfully applied to detection of target ions in living cells.

Porphyrin-magnetite nanoconjugates for biological imaging.

BACKGROUND: The use of silica coated magnetic nanoparticles as contrast agents has resulted in the production of highly stable, non-toxic solutions that can be manipulated via an external magnetic field. As a result, the interaction of these nanocomposites with cells is of vital importance in understanding their behaviour and biocompatibility. Here we report the preparation, characterisation and potential application of new "two-in-one" magnetic fluorescent nanocomposites composed of silica-coated magnetite nanoparticles covalently linked to a porphyrin moiety. METHOD: The experiments were performed by administering porphyrin functionalised silica-coated magnetite nanoparticles to THP-1 cells, a human acute monocytic leukaemia cell line. Cells were cultured in RPMI 1640 medium with 25 mM HEPES supplemented with heat-inactivated foetal bovine serum (FBS). RESULTS: We have synthesised, characterised and analysed in vitro, a new multimodal (magnetic and fluorescent) porphyrin magnetic nanoparticle composite (PMNC). Initial co-incubation experiments performed with THP-1 macrophage cells were promising; however the PMNC photobleached under confocal microscopy study. ß-mercaptoethanol (ß-ME) was employed to counteract this problem and resulted not only in enhanced fluorescence emission, but also allowed for elongated imaging and increased exposure times of the PMNC in a cellular environment. CONCLUSION: Our experiments have demonstrated that ß-ME visibly enhances the emission intensity. No deleterious effects to the cells were witnessed upon co-incubation with ß-ME alone and no increases in background fluorescence were recorded. These results should present an interest for further development of in vitro biological imaging techniques.

Pectic acid effects on prolactin secretion in GH3/B6 rat pituitary cell line.

BACKGROUND: Pectic acid extracted from plants increases the secretion of prolactin (PRL) when injected intravenously into ewes or fed to rats. Fragments of ewe hypophysis and lactating rabbit mammary gland incubated in vitro in the presence of pectic acid secreted more PRL and caseins compared to the controls. However, it is not known whether pectic acid directly stimulates PRL secretion in pituitary or interference of factors from hypophysis is required for this process. METHODS: GH3/B6 cells, a clonal strain of rat pituitary, were cultured and incubated with pectic acid (2.5-100 microg/mL). The integrity of cells was examined under pectic acid treatment microscopically. Controls or pectic acid treated cells were assayed for their ability to produce PRL. The PRL was assayed by Western-blotting and Radioimmunoassay. RESULTS: pectic acid did not have any significant effect on the viability of cells. After being incubated with pectic acid, the cells started to become circular and protuberant shape. The maximum stimulation and PRL secretion occurred at 100 microg/mL concentration within 30 min of incubation with pectic acid. CONCLUSION: pectic acid could stimulate the release of PRL in GH3/B6 cells in the short-term incubation. This result suggested that pectic acid is a non-toxic agent that could directly stimulate PRL secretion in pituitary cells without any interference of hypophysis.

Feedback from horizontal cells to rod photoreceptors in vertebrate retina.

Retinal horizontal cells (HCs) provide negative feedback to cones, but, largely because annular illumination fails to evoke a depolarizing response in rods, it is widely believed that there is no feedback from HCs to rods. However, feedback from HCs to cones involves small changes in the calcium current (I(Ca)) that do not always generate detectable depolarizing responses. We therefore recorded I(Ca) directly from rods to test whether they were modulated by feedback from HCs. To circumvent problems presented by overlapping receptive fields of HCs and rods, we manipulated the membrane potential of voltage-clamped HCs while simultaneously recording from rods in a salamander retinal slice preparation. Like HC feedback in cones, hyperpolarizing HCs from -14 to -54, -84, and -104 mV increased the amplitude of I(Ca) recorded from synaptically connected rods and caused hyperpolarizing shifts in I(Ca) voltage dependence. These effects were blocked by supplementing the bicarbonate-buffered saline solution with HEPES. In rods lacking light-responsive outer segments, hyperpolarizing neighboring HCs with light caused a negative activation shift and increased the amplitude of I(Ca). These changes in I(Ca) were blocked by HEPES and by inhibiting HC light responses with a glutamate antagonist, indicating that they were caused by HC feedback. These results show that rods, like cones, receive negative feedback from HCs that regulates the amplitude and voltage dependence of I(Ca). HC-to-rod feedback counters light-evoked decreases in synaptic output and thus shapes the transmission of rod responses to downstream visual neurons.

Proton-mediated feedback inhibition of presynaptic calcium channels at the cone photoreceptor synapse.

Generation of center-surround antagonistic receptive fields in the outer retina occurs via inhibitory feedback modulation of presynaptic voltage-gated calcium channels in cone photoreceptor synaptic terminals. Both conventional and unconventional neurotransmitters, as well as an ephaptic effect, have been proposed, but the intercellular messaging that mediates the inhibitory feedback signal from postsynaptic horizontal cells (HCs) to cones remains unknown. We examined the possibility that proton concentration in the synaptic cleft is regulated by HCs and that it carries the feedback signal to cones. In isolated, dark-adapted goldfish retina, we assessed feedback in the responses of HCs to light and found that strengthened pH buffering reduced both rollback and the depolarization to red light. In zebrafish retinal slices loaded with Fluo-4, depolarization with elevated K(+) increased Ca signals in the synaptic terminals of cone photoreceptors. Kainic acid, which depolarizes HCs but has no direct effect on cones, depressed the K(+)-induced Ca signal, whereas CNQX, which hyperpolarizes HCs, increased the Ca signals, suggesting that polarization of HCs alters inhibitory feedback to cones. We found that these feedback signals were blocked by elevated extracellular pH buffering, as well as amiloride and divalent cations. Voltage clamp of isolated HCs revealed an amiloride-sensitive conductance that could mediate modulation of cleft pH dependent on the membrane potential of these postsynaptic cells.

Nitric Oxide on the MMP-2 expression by human gingival fibroblasts / 대한치주과학회지

It has been suggested that increased number and activity of phagocytes in periodontitis lesion results in a high degree of reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, nitric oxide and peroxynitrite. There are few reports on the relationship between ROS and MMPs expressions in gingival fibroblast. We studied to elucidate whether and how ROS, especially nitric oxide affects the MMP expression. Human gingival fibroblasts and HT1080 cells (human fibrosarcoma sell line as reference) were grown in DMEM supplemented with 10 mM HEPES, 50 mg/L gentamicin, and 10% heat inactivated fetal bovine serum with addition of various reactive oxygen species (ROS). Culture media conditioned by cells were examined by gelatin zymography. HT1080 cells expressed proMMP-2 and proMMP-9, but human gingival fibroblasts (HGF) produced only proMMP-2. Hydrogen peroxide upregulated MMP-9 expression in HT1080 cells, whereas in human gingival fibroblast SNP treatment showed marked increase in MMP-2 level compared to other ROS. These results suggest that the effects of ROS on MMPs expressions are cell-type specific. RT-PCR for MMP-2 and TIMP-2 m-RNA were performed using total RNA from cultured cells under the influence various kinase inhibitors. In HT1080 cells, treatment with FPTI III (Ras processing inhibitor) and LY294002 (PI3-kinase inhibitor) resulted in inhibition of MMP-2 and MMP-9 expressions, suggesting that Ras/PI3-kinase pathway is important for MMPs expression in HT1080 cells. In gingival fibroblasts, treatment with FPTI III and PDTC (NFkB inhibitor) showed marked decrease in MMP-2 regardless of the of SNP, suggesting that Ras/NF-kB could be the key pathway for NO-induced MMP-2 expression in gingival fibroblasts. This study showed that ROS, especially nitric oxide, could be the critical mediator of periodontal disease progression through control of MMP-2 expression in gingival fibroblasts possibly via Ras/NF-kB pathway.

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro.

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2/HCO3- buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2/HCO3- (5%/26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (fCN) recorded. Increases in fCN evoked by ACh, ATP and NaCN in CO2- free saline were significantly reduced (P < 0.05, Wilcoxon test) when CO2/HCO3- was present in the superfusion medium. Thus, the presence of CO2/HCO3- buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation.

Medium requirements for neuritic outgrowth from goldfish retinal explants and the trophic effect of taurine.

The use of culture media of known composition are necessary for studying the role of trophic molecules. Since most of the in vitro research on regeneration of the optic nerve has been performed in the presence of fetal calf serum, the aim of this study was to obtain a medium in which the neuritic outgrowth from post-crush goldfish retinal explants could take place without adding fetal calf serum. After the lesion of the optic nerve (10 days), the retina of goldfish was dissected and explants were cultured for 5 and 10 days in the absence or in the presence of fetal calf serum, at which time the neuritic outgrowth was determined. Various concentrations and combinations of glucose, albumin, calcium, HEPES and taurine were used. The highest neuritic outgrowth was observed in the presence of fetal calf serum, in which condition the amino acid taurine increased length and density of neurites. Media supplemented with albumin, calcium or HEPES did not modify the outgrowth of neurites from the explants. However, glucose favored the neuritic outgrowth in a bell-shaped manner, although fibers were thinner than those observed in the presence of fetal calf serum. Taurine did not stimulate outgrowth of neurites from explants growing in a medium with optimal concentrations of glucose, indicating that elements of the fetal calf serum are determinant for the trophic effect of taurine. The present results contribute to further studies, such as those related to the effect of taurine and of trophic factors derived from the optic tectum, which would be performed in the presence of a medium free of fetal calf serum.

Effects of bicarbonate buffer on acetylcholine-, adenosine 5'triphosphate-, and cyanide-induced responses in the cat petrosal ganglion in vitro

Acetylcholine (ACh), adenosine 5'-triphosphate (ATP) and sodium cyanide (NaCN) activate petrosal ganglion (PG) neurons in vitro, and evoke ventilatory reflexes in situ, which are abolished after bilateral chemosensory denervation. Because in our previous experiments we superfused the isolated PG with solutions free of CO2 /HCO3¯ buffer, we studied its effects on the PG responses evoked in vitro. PGs from adult cats were superfused at a constant pH, with HEPES-supplemented (5 mM) saline with or without CO2 /HCO3¯ (5% / 26.2 mM) buffer, and carotid (sinus) nerve frequency discharge (âCN) recorded. Increases in âCN evoked by ACh, ATP and NaCN in CO2-free saline were significantly reduced (P<0.05, Wilcoxon test) when CO2 / HCO3¯ was present in the superfusion medium. Thus, the presence of CO2 / HCO3¯ buffer appears to reduce PG neurons sensitivity to ACh, ATP and NaCN, an effect that may underlie the lack of ventilatory reflexes after bilateral chemodenervation

Sustained in vivo activity of recombinant bovine granulocyte colony stimulating factor (rbG-CSF) using HEPES buffer.

The purpose of this study was to develop a long-acting injectable formulation of bG-CSF for veterinary use. However, in order to achieve sustained in vivo activity it was first necessary to stabilize the protein at the injection site. Preformulation studies, as well as literature, suggest that bG-CSF aggregates at neutral pH ranges (i.e., pH 6-8) and at temperatures of approximately 40 degrees C. Therefore, bG-CSF will not retain its activity for an extended period of time at the injection site. During this study we determined that HEPES buffer has a very significant impact on protein stability as well as on biological performance. Recombinant bovine granulocyte colony stimulating factor (rbG-CSF) was formulated in 1 M HEPES buffer for subcutaneous injection into cows. bG-CSF formulated in 1 M HEPES buffer resulted in sustained in vivo activity of bG-CSF compared to the "control" formulation (control formulation: 5% mannitol, 10 mM acetate buffer, 0.004% tween-80, pH 4). White blood cell (WBC) count was used as a marker to evaluate in vivo activity of the formulation. WBC numbers remained above a threshold value for only 24-30 h for the control formula. However, when bG-CSF was formulated in 1 M HEPES, the WBC remained above threshold for 3 days or 72 h. Formulating bG-CSF in 1 M HEPES at pH 7.5 also resulted in greater solution stability. This was surprising since bG-CSF is intrinsically not stable at neutral pH. The effect of 1 M HEPES on the T(M) (temperature at maximum heat flow on calorimetry scan) of bG-CSF was determined by microcalorimetry. In the absence of 1 M HEPES buffer the T(M) was 48 degrees C (onset approximately 40 degrees C), while bG-CSF formulated in 1 M HEPES buffer has a T(M) of 59 degrees C (onset approximately 50 degrees C). Similar organic buffers, such as MOPS, HEPPS, TES, and tricine, also resulted in improved solution stability as well as in sustained in vivo activity. The dramatic effect of these buffers on stability and biological performance of bG-CSF is not well understood. One hypothesis is that the electrostatic interaction between the zwitterionic form of these buffers and bG-CSF provides stabilization against denaturation.

Effect of tooth-bound fluoride on enamel demineralization/ remineralization in vitro.

The effect of tooth-bound fluoride (F) on enamel caries formation was investigated under the condition that loosely bound F was essentially absent. Eighteen thin enamel sections, prepared from the lingual or buccal surfaces of extracted human molars, were embedded in acrylic resin with the enamel surfaces exposed. The sections were placed in a pH 7 remineralizing solution (RS; 1.2 mmol/l Ca, 0.72 mmol/l P, 30 mmol/l KCl, 50 mmol/l HEPES) for 5 days, and were randomly divided into 3 groups: (1) control group that received no treatment, (2) acidulated phosphate fluoride (APF) group that received 5 cycles of a 4 min treatment with APF gel followed by immersion in the RS for 2 days (RS changed daily) and (3) dicalcium phosphate dihydrate (DCPD) - APF group that received 5 cycles of a 4-min pH 2.1 DCPD-forming solution followed by 4 min APF gel and then placed in the RS for 2 days. After the treatment cycles, the sections were washed in a constant composition F titration system to remove loosely bound F. An in vitro model, which consisted of cycles of de- (6 h) and remineralization (18 h) each day for 5 days, was used to produce caries-like lesions in the specimens. The DeltaZ (mineral loss) values, measured by quantitative microradiography, of the lesions formed in the three groups were (mean +/- standard deviation; n = 6) 91.2+/-12.3 microm for the control group, 41.3+/-10.1 microm for the APF group and 21.2+/-4.8 microm for the DCPD-APF group. The same system produced lesions in untreated shark enamel with a mean DeltaZ of 4.4+/-0.3 microm (n = 12). One-way fixed-effects ANOVA indicated that mineral loss was significantly different among the different groups (p<0.05). The results showed that enamel resistance to lesion formation increased with increasing tooth-bound F content. Shark enamel was much more resistant to demineralization than human enamel.

Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes.

The present study evaluated the effects of various components in a chemically defined medium on the development of IVM/IVF porcine embryos. The investigated components included energy substrates (lactate, pyruvate or glucose, alone or in various combinations), amino acids (glutamine, glycine or alanine), PVP and HEPES buffer. The effects of each energy substrate were the same as the control. However, a mixture of lactate with either of the other energy substrates increased the development rate. Glutamine tended to decrease rate of the development more than other amino acids, and this inhibition was dose dependent. Both PVP and HEPES buffer did not affect development rate. However, more than 35 mM HEPES buffer induced fragmentation From the above results, a new culture medium was designed (supplemented with 0.276 mM glycine, 0.176 mM alanine, 15 mM HEPES buffer and 1% (wt/vol) PVP in BSA-free Whitten's medium with or without glucose). The new medium resulted in a higher embryo development rate (20.4 and 16.3%) than that obtained with the control medium (10.0%).

Role of ATF on Transcriptional Regulation of DNA Topoisomerase II a Gene in HL - 60 Arrested to G2 / M and M Phase / 대한암학회지

PURPOSE: To gain insight on transcriptional repression of Topo II a in HL-60 cells arrested to G2/M and M phase, the levels of Topo IIa mRNA and the binding activity of ATF have been investigated with Northern blot hybridization and DNA mobility shift assay, respectively. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-mactivated fetal bovine serum and antibiotics in a humidified 5% CO2 at 37C degree. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. A Xho I-Mlu I fragment of phTOP2 was used as probe for Northern blot analysis of Topo II a mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA oligomer (upper strand, 5-TCTCCGCTATGACGCCGAGTGGTG-3) for ATF binding activity was mixed with nuclear extracts in a 20 pl reaction volume containing 60 mM KC1, 12 mM HEPES, pH 7.9, 5 mM MgCl2, 0.2 mM EDTA, 0.2 mM DTT, 12% glycerol, and 2 ug of poly [dI-dC]. RESULTS: HL-60 cells were arrested at G2/M phase and M phase after taxol or nocodazole treatment. The levels of Topo II a mRNA were reduced at 24 hours after exposure with nocodazole or taxol but the unknotting activities were not changed. DNA mobility shift assay using oligonucleotide containing the ATF binding site showed that ATF binding activity was reduced after pretreatment of nododazole or taxol. CONCLUSIONS: These results suggest that the reduction of ATF binding activity may be important to transcriptional repression of Topo II a gene by nocodazole and taxol in HL- 60 cells.

The effect of intrinsic fluoride in cows' milk on in vitro enamel demineralization.

The fluoride concentration in cows' milk has been reported to vary with the fluoride levels in drinking water but it seldom exceeds 0.5 microg/ml. This raised a question as to whether any caries-protective effect could be attributed to the intrinsic fluoride of milk. Two samples of cows' milk with intrinsic fluoride concentrations of 0.03 and 0.3 microg/ml, respectively, were assessed for their protective effect on enamel in an in vitro demineralization model at relatively severe and mild acidic challenges (pH 4.6 and 5.0, respectively). Polished enamel discs were incubated individually in 5.0 ml of demineralization solution for 20 h per day alternated with 1-hour incubations in 1.0 ml of milk or control buffers: group 1, demineralization solution only (negative control); group 2, milk with 0.03 microg/ml fluoride; group 3, milk with 0.03 microg/ml fluoride; supplemented with NaF to 0.3 microg/ml fluoride; group 4, milk with 0.3 microg/ml fluoride; group 5, 0.3 microg/ml fluoride in 20 mM HEPES, pH 6.7; group 6, milk with 0.03 microg/ml fluoride supplemented with NaF to 5.0 microg/ml fluoride (positive control). The solutions were renewed each day and the calcium concentration in the demineralization solutions was followed during 4 days. The results showed that the protective effect of intrinsic milk fluoride on enamel is limited by the severity of the acidic challenge: There was a significant inhibition of the demineralization in groups 3-6 compared to groups 1 and 2, but only at pH 5.0 (p<0.0001) and not at pH 4.6 (p = 0.2). The organic components of milk had limited protection against demineralization because milk and HEPES with the same fluoride concentration gave similar results. The 36% reduction in calcium loss at pH 5.0 by treatment with milk with only 0.3 microg/ml fluoride is an indication that intrinsic milk fluoride has some caries-protective properties.

Transcriptional REpression of Vimentin Gene During All-TTrans Retinoic Acid-Induced Differentiation of HL-60 Cells / 대한내분비학회지

BACKGROUND: Vimentin is the major intermediate-size filament in the cytoplasm of cells from mesenchymal origin. The HL-60 cell is a unique human leukemic cell line capable of terminal differentiation with several chemical inducers, and then the cell line becomes a fre#quently described model system for cell differentiation in vitro. Vimentin mRNA is reduced during all-trans retinoic acid (retinoic acid) -dependent differentication but increased by 12-0-tetradecanoylphorbol-13-acetate (TPA). In this paper, we have investigated on the mechanism of transcriptional repression of vimentin gene during retinoic acid-dependent differentication of HL-60 cell. METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics in a humidified 5% CO at 37C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe (Upper strand, 5-CGCITGATGAGTCAGCCG-3) for AP-1 binding activity was mixed with nuclear extracts in a 20 pL reaction volume containing 300 mM KCI, 60 mM HEPES, pH 7.9, 25mM MgC1, 1mM EDTA, 1mM DTT, 60% glycerol, and 2 pg of poly[dI-dC]. RESULTS: The level of vimentin mRNA was decreased at 12 hours after retinoic acid treatment, and not detected at 48 hours. The level of vimentin mRNA was reduced in proportion to concentration of retinoic acid, Retinoic acid-reduced vimentin mRNA was no change in cells treated with cycloheximide. Retinoic acid-dependent decrease of vimentin mRNA was partially recovered by staurosporin pretreatment. In DNA mobility shift assay, AP-1 binding activity was reduced at 48 hr during retinoic acid-induced differentiation. CONCLUSION: These results suggest that the transcriptional repression of vimentin gene during retinoic acid-induced differentiation in HL-60 cells is correlated with reduction of DNA binding activity of AP-1.

Role of AP-1 Transcriptional Regulation of Vimentin Gene during 12-O-Tetradecanoylphorbol-13-Acetate Differentiation of HL-60 Cells / 대한암학회지

PURPOSE: To gain insight on the role of AP-1 in transcriptional regulation of vimentin gene during differentiation of HL-60 cells by 12-0-tetradecanoylphorbol-13-acetate (TPA), the levels of vimentin mRNA and AP-1 have been investigated with Northern blot hybridization and DNA mobility shift assay. MATERIALS AND METHODS: HL-60 cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum and antibiotics in a humidified 5% CO2 at 37 degree C. Total RNA was prepared by a modification of the method of Karlinsey et al. Northern blot hybridization was performed by the method of Virca et al. EcoRI fragment of pVIM-GEM was used as probe for vimentin mRNA. DNA mobility shift assay was performed by the method of Lim et al. End labeled DNA probe(Upper strand, 5'-CGCTTGATGAGTCAGCCG- 3') for AP-1 binding activity was mixed with nuclear extracts in a 20 microliter reaction volume containing 300 mM KC1, 60 mM HEPES, pH 7.9, 25 mM MgCl2, 1 mM EDTA, 1 mM DTT, 60% glycerol, and 2 microgram of poly[dI-dC]. RESULTS: TPA increased vimentin mRNA levels, with maxima1 stimulation reached at 24 hr. The level of vimentin mRNA was induced in proportion to the concentration of TPA. TPA-induced vimentin mRNA was almost reduced by actinomycin-D pretreatment. TPA- induced stimulation of vimentin gene was completely reduced by staurosporin pretreatment. In DNA mobility shift assay, AP-I newly appeared at 24 hr during TPA- induced differentiation and was almost not detected after the pretreatment of staurosporin. CONCLUSIONS: These results suggest that the induction of vimentin mRNA during TPA- dependent differentiation in HL-60 cells may be mediated by protein kinases C signal transduction and AP-1 is important to transcriptional regulation.

Effect of amino acids and alpha-amanitin on the development of rabbit embryos in modified protein-free KSOM with HEPES.

Four experiments were conducted to test the effects of Eagle's non-essential amino acids (NEAA) and essential amino acids (EAA), glycine, and the RNA polymerase inhibitor, alpha-amanitin, on the development of preimplantation rabbit embryos in modified protein-free KSOM medium. Embryos were distributed randomly into different treatments and cultured in 5% O2:5% CO2:90% N2. In experiment 1, 100% of the embryos became blastocysts in the medium with Eagle's 1X NEAA and 0.5X EAA, but 100% stopped development at the morula stage in KSOM without amino acids. These morulae failed to develop further when transferred to amino acid supplemented medium after 72 hr of culture. Glycine alone in modified KSOM (experiment 2) was ineffective in supporting development of 8-16-cell stage embryos past the morula stage. In experiment 3, the addition of 1X NEAA and 0.5X EAA at 0, 12, 24, 36, and 48 hr of culture resulted, respectively, in 57, 65, 65, 44, and 14% blastocysts on Day 3 (P < 0.05) and 86, 77, 77, 78, and 69% on Day 5 (P > 0.05). Omission of Eagle's amino acids until 48 hr clearly delayed embryo development. In experiment 4, when alpha-amanitin (20 microM) was added to the medium containing Eagle's amino acids after 0, 12, 24, 36, and 48 hr of culture most embryos cleaved only once or twice after adding the alpha-amanitin. Without the inhibitor, 94% of the zygotes developed into blastocysts. These results indicate that modified KSOM or KSOM plus glycine could not support rabbit embryo development past the morula stage, but this block was overcome by adding Eagle's amino acids. An exogenous source of amino acids was not critical for embryo development during the first 24 hr of culture, but was required after that for development to equal controls. Addition of alpha-amanitin at multiple pre-blastocyst stages limited further embryo development to one or two cleavage divisions, with no blastocyst development.

In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media.

The objective was to establish an in vitro system in which bovine oocytes can be matured, fertilized, and cultured up to the blastocyst stage without support of serum, BSA, or somatic cells. Media consisted of modified tissue culture medium 199 (mTCM 199) with ovine LH (oLH) for maturation (IVM), experimental alterations of modified defined medium (mDM) for sperm selection and insemination (IVF), and citrate+synthetic oviductal fluid+nonessential amino acids (c-SOF+NEA) for zygote/embryo culture (IVC). Effects of heparin, BSA, polyvinyl alcohol (PVA), penicillamine (P), Hepes, and sodium bicarbonate (NaHCO3) were studied. Results included proportions of oocytes that cleaved by 48 h and that reached morulae by 120 h, blastocysts by 168 h, and expanded blastocysts by 216 h postinsemination (pi). Best results were obtained when the IVF medium included 0.5 mg P+1.0 mg PVA per milliliter with no more than 10 mM Hepes, and when 3.0 mg PVA/ml and 10 mM Hepes were present for IVC. Different concentrations of NaHCO3, up to 50 mM from 25 mM, during IVF did not alter results. Embryos produced in defined conditions yielding the best results remained viable after vitrification as evidenced by continued development in vitro for 96 h postthawing. Bovine oocytes matured in defined medium supplemented with LH were fertilized and cultured up to the blastocyst stage in chemically defined conditions that afforded results comparable to those reported earlier after inclusion of serum, BSA, and/or somatic cells.