SLPI knockdown induced pancreatic ductal adenocarcinoma cells proliferation and invasion.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease and still continues to have the worst prognosis of all gastrointestinal malignancies. Reports have demonstrated that secretory leukocyte protease inhibitor (SLPI) is overexpressed in various cancers and may be a potential therapeutic strategy for the treatment of different cancers. However, the possible role of SLPI in PDAC is still unknown. In the present study, we investigate the effects of SLPI gene knockdown on the biological behavior of human pancreatic cancer cells. The expressions of SLPI were detected, by qRT-PCR and Western blot, in human PDAC tissues as well as AsPC-1, BxPC-3 and PANC-1 cells. After transfection with siRNA targeting to SLPI, SLPI expression was detected by qRT-PCR and Western blot in cells. Cell proliferation and apoptosis were also evaluated by MTT assay and flow cytometry (FCM). The trans-well assays were also employed to explore the effects of SLPI knockdown on the migration and invasion of PDAC cells in vitro. RESULTS: The expressions of SLPI derived from human PDAC and PDAC cell lines were significant higher than those of control groups, respectively (P < 0.05). Regression analysis showed elevated SLPI level was positive correlated with development of PDAC. The siRNA target to SLPI significantly decreased the expressions of SLPI in these PDAC cell lines. Following SLPI-siRNA transduction, the proliferative capacity of the AsPC-1, BxPC-3 and PANC-1 cells was significantly inhibitions, compared to the blank (PDAC-wild type cells) and negative (non-targeting scrambled siRNA transduced PDAC cells) control ones, respectively (P < 0.05). Moreover, SLPI knockdown significantly increased the apoptosis fractions and reduced the migration and invasion of PDAC cells in vitro (P < 0.05). CONCLUSIONS: The present study demonstrated that: i) SLPI played an important role in PDAC progression; ii) SLPI might be an important characteristic of malignant PDAC associated with migration and invasion in vitro; and iii) siRNA targeting to SLPI might be a potential therapeutic strategy for the treatment of PCC.

A boron-containing carbazole dimer: synthesis, photophysical properties and sensing properties.

A novel boron-containing π-conjugated compound has been synthesized by the introduction of electron-acceptors (dimesitylboron groups) at the 3,3'-positions of a carbazole dimer (electron-donor). The compound possesses excellent electrochemical properties and high fluorescence quantum yields. In addition, is a sensitive fluorescence sensor with remarkable colour changes and the results could be confirmed through theoretical calculations of the compounds and [(n)Bu(4)N](+)(2)[·(F)(2)](2-). Our studies indicate that could be used as an excellent optoelectronic material in OLED devices and a ratiometric fluorescent chemosensor.

Coordination reactions of 2-pyridinecarboxaldehyde-phenylhydrazonatolithium with selected transition metal (Zn, Sn, Fe, Co, Ni and Zr) chlorides and its coupling reaction with dichloromethane.

The reactions of 2-pyridinecarboxaldehyde-phenylhydrazonatolithium C5H4Npy-CH[double bond, length as m-dash]Ni-NaLi(Ph) (abbreviated as Li) with a 1/2 equivalent of anhydrous metal (Zn, Sn, Fe and Co) chlorides or NiCl2(DME) (DME = 1, 2-dimethoxyethane) produced the corresponding mononuclear metal(ii) complexes ( and ), in which each ligand acts as a bidentate ligand and the coordination geometries around the metals are shown to be tetrahedral within the complexes , , and , respectively, and a tetragonal pyramid in the complex . The reaction of Li successively with sodium tert-butoxide and anhydrous ZrCl4 afforded the unanticipated bizirconium complex , in which each monoanionic ligand behaves as a tridentate bridge. Whereas treatment of Li with NiCl2 and then CH2Cl2 led to an interesting methylene-bridged bis(2-pyridyl-phenylhydrazone) compound in moderate yield; a comparative experiment showed that when the Li reacted with CH2Cl2, the coupling compound was also obtained but in very low yield. A plausible mechanism of compound formation was also proposed and supported by the density functional theory (DFT) calculations. All the synthesized compounds were characterized by single-crystal X-ray diffraction.

Effect of cadmium on cytogenetic toxicity in hairy roots of Wedelia trilobata L. and their alleviation by exogenous CaCl2.

Effects of cadmium (Cd) alone and in combination with calcium on mitosis and chromosomal aberration in the hairy root tips of Wedelia trilobata were investigated. The results showed that Cd concentrations below 50 µmol/L had a lesser or even a promoting effect on the mitotic index (MI) and the rate of chromosomal aberration in hairy root tips, while those higher than 100 µmol/L significantly decreased the MI and gradually stimulated the rate of chromosomal aberrations with prolonged time and increasing concentrations of Cd. Concentrations of 50 µmol/L Cd mainly induced C-mitosis, while more than 100 µmol/L Cd mainly caused chromosome breakage and chromosome adhesion in hairy root tip cells. When cultured with 300 µmol/L Cd, micronuclei were only observed in the interphase, middle, and late phase of hairy root tip cells. Compared with untreated controls, exogenous calcium had an alleviating effect on Cd-induced cytotoxicity by effectively enhancing the MI and reducing the rate of chromosomal aberration in root tip cells. The results presented here provide evidence that W. trilobata hairy roots with rapid autonomous growth could be used as a sensitive tool for monitoring and evaluation of Cd pollution in the environment.

Aggregation induced ratiometric fluorescence change for a novel boron-based carbazole derivative.

Nanoparticles of a novel boron-based carbazole derivative have been reported. They exhibit efficient green fluorescence, aggregation induced ratiometric fluorescence change and green/blue fluorescent switching to sense VOCs.

Synthesis, photophysical and electrochemical properties of a carbazole dimer-based derivative with benzothiazole units.

A novel A-π-D-π-D-π-A type compound, containing two benzothiazole rings as electron acceptors and two N-ethylcarbazole groups as electron donors, (E)-1,2-bis(3-(benzothiazol-2-yl)-9-ethylcarbazol-6-yl)ethene (BBECE), was synthesized and characterized by elemental analysis, NMR, MS and thermogravimetric analysis. Electrochemical property of compound BBECE was studied by cyclic voltammetry analysis. The absorption and emission spectra of BBECE was experimentally determined in several solvents and simultaneously computed using density functional theory (DFT) and time-dependent density functional theory (TDDFT). The calculated absorption and emission wavelengths are coincident with the measured data. The lowest-lying absorption spectra can be mainly attributed to intramolecular charge transfer (ICT), and the fluorescence spectra can be mainly described as originating from an excited state with intramolecular charge transfer (ICT) character. The molecular orbitals (HOMO and LUMO), the ionization potential (IP), the electron affinity (EA) and reorganization energy of compound BBECE were also investigated using density functional theory (DFT). The results show that compound BBECE exhibited excellent thermal stability and electrochemical stability as well as high fluorescence quantum yield, indicating its potential applications as an excellent optoelectronic material in optical fields.

Experimental and theoretical study of two new pyrazoline derivatives based on dibenzofuran.

Two novel pyrazoline derivatives, named 2,8-bis(1,3-diphenyl-pyrazoline-5-yl)dibenzofuran (A) and 2,8-bis(1-(4-bromophenyl)-3-phenyl-pyrazoline-5-yl)dibenzofuran (B), were synthesized and characterized by elemental analysis, NMR, MS and thermogravimetric analysis. The absorption and emission spectra of them were determined by experimental methods in different polar solvents and were computed using the density functional theory (DFT) and the time-dependent density functional theory (TDDFT) at the same time. The calculated absorption and emission wavelengths are in good agreement with the experimental data. The fluorescence quantum yields and fluorescence lifetimes of them in different polar solvents were studied by means of steady state and time resolved fluorescence. The calculated reorganization energy for hole and electron indicates that the two compounds are in favor of hole transport than electron transport. The results show the two compounds present high fluorescence quantum yields and excellent thermal stability. It makes them of great interest as novel fluorescent probes and optoelectronic materials.

Experimental and theoretical study of three new benzothiazole-fused carbazole derivatives.

Three new D-π-A type compounds, each containing one benzothiazole ring as an electron acceptor and one N-ethylcarbazole group as electron donor, were synthesized and characterized by elemental analysis, NMR, MS and thermogravimetric analysis. The absorption and emission spectra of three compounds were experimentally determined in several solvents and were simultaneously computed using density functional theory (DFT) and time-dependent density functional theory (TDDFT). The calculated reorganization energy for hole and electron indicates that three compounds are in favor of hole transport than electron transport. The calculated absorption and emission wavelengths are well coincident with the measured data. The calculated lowest-lying absorption spectra can be mainly attributed to intramolecular charge transfer (ICT). And the calculated fluorescence spectra can be mainly described as originating from an excited state with intramolecular charge transfer (ICT) character. The results show that three compounds exhibited excellent thermal stability and high fluorescence quantum yields, indicating their potential applications as excellent optoelectronic material in optical field.

Experimental and theoretical study of a new carbazole derivative having terminal benzimidazole rings.

A novel intramolecular donor-acceptor compound has been synthesized and characterized. This compound was a symmetrical A-pi-D-pi-A type molecule containing two benzimidazole rings as two electron acceptors (A) and an N-ethylcarbazole group as electron donors (D). The absorption and emission spectra of the compound were determined by experimental methods in solution and were computed by using the density functional theory (DFT) and the time-dependent density functional theory (TDDFT) in gas phase and in chloroform solution. The calculated absorption and emission wavelengths were in good agreement with the experimental ones. The fluorescence quantum yields and fluorescence lifetimes of the compound in several solvents have been studied by means of steady state and time resolved fluorescence. The results showed the compound had high quantum yield. The cross-section of two-photon absorption (TPA) of the compound was measured by using femtosecond laser in dichloromethane solution. The result indicated the cross-section maximum of two-photon absorption was 430 GM at 600 nm. These results made the compound of great interest as a new fluorescent probe and photoluminescence material.

[Induction and in vitro culture of hairy roots of Solanum nigrum L. var. pauciflorum Liou and its solasodine production].

By using genetic transformation of Agrobacterium rhizogenes and liquid culture, induction and culture conditions for Solanum nigrum L. var. pauciflorum Liou hairy roots and its solasodine production and consumption changes of N resource and calcium in the medium during liquid culture were investigated. The results showed that hairy roots could be initiated from the cut edges of leaf explants 5 days after inoculation with the strain of A. rhizogenes ATCC15834. The percentage of rooted leaf explants 25 days after infection was more than 90%. Hairy roots could grow rapidly on solid or liquid growth regulator-free MS medium. The PCR amplification of rol genes and virC gene showed that rol genes of Ri plasmid of A. rhizogenes were integrated in the genome of transformed hairy roots of S. nigrum L var pauciflorum. The hairy roots could produce medicinal secondary metabolites solasodine and the amount of solasodine in the hairy root cultures reached a level of 582.05 microg/g dry weight and was 1.31 times as much as those in the untransformed roots. The hairy roots grew very slowly in 0-5 days in the liquid medium, then, very fast from 5 to 15 days. During liquid culture, NO3(-) and NH4(+) in the medium were gradually absorbed and utilized by hairy roots. NH4(+) -N of the medium was used up at day 15 of the culture, while NO3(-) in the medium was not used up, the content of which was 44.7% of the initial amount. Ca2+ of the medium was gradually absorbed and utilized during liquid culture and it was not used up at day 25, the content of which was still 43.5% of the initial amount. The results presented here had provided the possibilities on how to prepare optimum medium for large scale cultivation and production of solasodine from S. nigrum L. var. pauciflorum hairy roots.

Expression of recombinant Digitalis lanata EHRH. cardenolide 16'-O-glucohydrolase in Cucumis sativus L. hairy roots.

The coding sequence for the Digitalis lanata EHRH. cardenolide 16'-O-glucohydrolase was inserted downstream of the 35S promoter in the binary vector pBI121 resulting in plant expression vector pBI121cgh. Cotyledon explants excised from 10-day-old seedlings of Cucumis sativus L. were transformed using Agrobacterium rhizogenes 15834 harbouring pBI121cgh. Hairy roots were obtained from infected cotyledon explants in vitro 10 days after inoculation. PCR amplification of coding sequences for cgh I, rolB and rolC from Ri plasmid showed that the aimed sequences were inserted into the genome of transformed cucumber hairy roots. Glycolytic activity of the transgenic CGH I was measured by HPLC using Lanatoside glycosides as substrate. Therefore, the cgh I transformed cucumber hairy roots may provide a valuable model for biotransformation of natural compounds by recombinant enzymes.

[Induction of cucumber hairy roots and effect of cytokinin 6-BA on its growth and morphology].

Hairy roots of Cucumis sativus L. could be incited directly from the cut edges of 10-day-old cotyledon explants after infection with the strain Agrobacterium rhizogenes ATCC15834 harboring agropine-type plasmid, pRiA4b for 5 days. It was observed that the percentage of rooting cotyledon explants was more than 90 % 10 days after infection. Hairy roots could grow rapidly and highly branched on solid plant growth regulator-free MS medium. The PCR amplification of rol B genes and vir C gene showed that T-DNA of Ri plasmid of A. rhizogenes was integrated and expressed into the genome of transformed cucumber hairy roots. A bacterium-free transformed cucumber hairy root line was selected to culture on solid MS medium to examine influence of exogenous cytokinin 6-BA on growth and morphology alteration of cucumber hairy roots. The results showed that cytokinin 6-BA can influence the growth and altered the morphology of hairy roots. With increasing of 6-BA concentrations, Cucumber hairy roots become shorter and thicker and less branched. 6-BA at the concentration of 0.1 to approximately 3.0 mg/L could delay the appearance of maximum growth peak by 5 days and decreased the content of soluble protein, enhanced the activities of SOD and POD and decreased the levels of endogenous ethylene evolution in cucumber hairy roots. Our results also indicated that cytokinin 6-BA in the medium could influence growth and morphology alternation of cucumber hairy roots and delay its senescence of hairy roots by acting through ethylene.

[Effects of sucrose and light on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots].

Effects of sucrose concentrations and light on the growth and production of total isoflavones and puerarin in Pueraria phaseoloides hairy roots cultured onto solid MS media supplemented with 1%, 3%, 5%, 7% and 9% sucrose, respectively, were investigated. The results showed that among the sucrose concentrations tested, 3% sucrose in the medium enhanced the growth and stimulated accumulation of total isoflavones and puerarin in P. phaseoloides hairy roots, After cultured for 20 days, the biomass of hairy roots reached 0.48 g (DW dry weight)/flask and its contents of total isoflavones and puerarin were 25.44 mg/g (DW) and 11.64 mg/g (DW), respectively. In comparison with 3% sucrose, the dry weight proliferation of hairy roots cultured with 5% sucrose was increased by 7.0%, while cultured with 1%, 7% and 9% sucrose, the dry weight proliferation of hairy roots was decreased by 62.4%, 42.8% and 65.3%, their total isoflavones content was decreased by 57.4%, 13% and 33.4% and their puerarin content was decreased by 47.9%, 15.8% and 35.1%; but their content of total soluble sugars was increased 0.52, 1.45 and 1.54 times, respectively. Compared with hairy roots in blue light and white light, the biomass of hairy roots cultured in the dark for 30 days was 0.83 g (DW)/flask and was increased by 37.1% and 23.3%, respectively. The content of total isoflavones in hairy roots cultured in white light was as much as 1.15 times and 1.19 times that in blue light and in the dark, respectively. It was also observed that hairy roots cultured in blue light and white light partly became light green and that blue light could inhibit accumulation of puerarin in hairy roots and the puerarin content in hairy root cultured in white light and in the dark were 1.61 times and 1.52 times that in blue light, respectively.

[Induction of hairy roots of Pueraria phaseoloides and its culture in liquid and solid medium].

An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides, which contains puerarin and daidzein with hypothermic, spasmolytic, hypotensive and anti-arrhythmic activities, by using agropine-type Agrobacterium rhizogenes ATCC 15834 was developed. Hairy roots could be obtained directly from the cut edges of petioles of leaf explants of P. phaseoloides or via callus 20 days after inoculation with agrobacterium. The percentage of rooted leaf explants 35 days after infection was about 85%. Hairy roots could have a rapid growth on solid or liquid growth regulator-free MS medium. The transformation of hairy roots was confirmed by PCR amplification of rol B and rol C genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture, the biomass (fresh weight and dry weight), the reactive oxygen species (ROS) and the total content of soluble sugar in hairy roots cultured for 15 days in solid and liquid medium were detected, respectively, by the method of fluorescence labeling of 2',7'-dichlorofluorescein diacetate (2',7'-DCFH-DA) and by the anthrone colourimetry. Compared to hairy roots in solid medium, hairy roots grew more rapidly in liquid medium but formed no callus and appeared to become brown earlier during culture. The fresh weight, the dry weight, the total content of soluble sugar and the levels of reactive oxygen species of hairy roots cultured into liquid medium MS without plant growth regulators for 15 days were 1.59 times, 1.18 times, 5.25 times and 1.16 times, respectively as much as that of hairy roots cultured onto solid medium. Our results firstly indicate that P. phaseoloides hairy roots in solid medium can utilize or metabolize more soluble sugar but produce less reactive oxygen species than that in liquid medium. This may be related to the fact that hairy roots are easier to turn brown in liquid medium than that onto solid medium. Our results have laid a foundation for defining optimum culture manner for large-scale cultivation and large-scale production of secondary metabolites of P. phaseoloides hairy roots.

[Induction and culture of hairy boots in Phytolacca esculenta and its saponin production].

Hairy roots appeared in vitro 20 days after inoculation of Phytolacca esculenta leaf explants with the strain of Agrobacterium rhizogenes R1601. The frequency of leaf explants transformed by R1601 was up to 70%. Hairy roots can be incited directly from the veins of explants or via callus. Hairy roots induced by R1601 grew rapidly on medium MS without hormone and were 85.6% higher in respiration rate than control roots. Transformation was confirmed by opine detection and the amplification of rol B and rol C genes from the hairy roots of P. esculenta. The total saponin content in hairy roots of P. esculenta was about 1.54 times as much as in natural roots whereas polysaccharides content was about 70% times as much as in the natural roots.

[Effect of sucrose concentration on the growth and production of secondary metabolites in Pueraria phaseoloides hairy roots].

Effects of sucrose concentrations on the growth and production of puerarin and isoflavones compounds in Pueraria phaseoloides hairy roots induced by Agrobacterium rhizogenes ATCC15834 were investigated. Changes of sucrose consumption in the medium during liquid culture were also determined. The results showed cultured for 16 days in MS medium with 5%, 4%, 3% and 2% sucrose, the proliferation times of dry weight of hairy roots were 11.7, 11.9, 10.1 and 5.9, respectively. 3% sucrose concentration in liquid medium was the best for accumulation of puerarin and isoflavones in the hairy roots. The highest content of puerarin, 5.147 mg/g DW, was obtained after 12 days of liquid culturing while the highest content of isoflavones, about 27.76 mg.g.DW, was gained after 16 days in culturing. Sucrose concentration decreased as hairy root growth proceeded. The growth rate and the content of soluble sugar in hairy roots of P. phaseoloides was directly proportional to the rate of sucrose utilization in the liquid medium during the whole culture. It was observed that the highest content of soluble sugar in hairy roots was at day 12 of liquid culture and sucrose in the liquid medium was used up at the end of 16 days of culture.

[Culture of hairy roots in Pueraria phaseoloides and its puerarin production].

An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides was developed, by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained via callus from the cut edges of leaf explants of P. phaseoloides 20 days after inoculation with agrobacterium. 35 days after infection, the percentage of rooted leaf explants was about 85%. Hairy roots could have an autonomous growth on solid or liquid growth regulator-free MS medium but grew more rapidly and formed no callus during culture in liquid medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture, the mitochondrial membrane potential in hairy roots cultured for 15 days in solid and liquid medium were also detected by the method of fluorescence labeling of 3,3'-dipropylthiadicarbocyanide iodide. The results showed mitochondrial membrane potential of hairy roots in liquid medium was 11.8 times higher than that on solid medium. The content of puerarin in hairy roots reached a level of 1.190 mg/g.dry.wt and was 2.5 times as much as that in the roots of P. phaseoloides seedlings and was also 1.067 times as much as that in the crude drug of several year-old Pueraria roots. Our experiments have laid a foundation for large-scale production of puerarin in P. phaseoloides hairy roots.

[Benzylbis(dimethylamino)methylsilyl-kappa2C,N](N,N,N',N'-tetramethylethylenediamine-kappa2N,N)lithium(I).

The title compound, [Li(C12H21NSi)(C6H16N2)], is an intermediate in the synthesis of the corresponding organometallic compounds. The molecule has an unusual C-Si-N-Li four-membered heterocycle which adopts a folded conformation, with the coordination around the Li, N, C and Si atoms being distorted tetrahedral. Its structure is strongly supported by 1H NMR, 13C NMR and 13C-1H correlation spectra. The compound has potential for application in the synthesis of other novel organometallic compounds.

Scale-up production of puerarin from hairy roots of Pueraria phaseoloides in an airlift bioreactor.

Hairy roots of the Chinese herb, Pueraria phaseoloides, obtained from leaf explants and transformed with the Agrobacterium rhizogenes, were cultured in 2.5 l airlift bioreactors for three weeks. Puerarin accumulated at 5,570 microg g(-1) dry wt, which is near 200 times as much as in 250 ml flask cultures. In addition, puearin was exuded into the nutrient medium at final concentrations higher than in the hairy roots themselves.