Nonlinear Optical Properties of Large-Sized Gold Nanorods

The nonlinear optical properties of single gold nanorods (GNRs) with a large diameter of ～200 nm and a long length of ～800 nm were investigated by using a focused femtosecond (fs) laser light with tunable wavelength. While the linear and nonlinear optical properties of small-sized GNRs have been extensively studied, the nonlinear optical properties of large-sized GNRs and the effects of high-order surface plasmon resonances remain unexplored. Second harmonic generation (SHG) or/and two-photon-induced luminescence (TPL) were observed in the nonlinear response spectra, and their dependences on excitation wavelength and polarization were examined. The scattering and absorption spectra of the small- and large-sized GNRs were compared by using the discrete dipole approximation method. It was found that the extinction of large-sized GNRs is dominated by scattering rather than absorption, which is dominant in small-sized GNRs. In addition, it was revealed that the excitation wavelength-dependent SHG of a GNR is governed by the linear scattering of the GNR and the maximum SHG is achieved at the valley of the scattering spectrum. In comparison, the excitation wavelength dependence of TPL is determined by the absorption spectrum of the GNR. The polarization-dependent SHG of a GNR exhibits a strong dependence on the dimension of the GNR, and it may appear as bipolar distributions parallel or perpendicular to the long axis of the GNR or multipole distributions.     

负载雷公藤甲素的TPGS-b-(PCL-ran-PGA)纳米制剂体外抑制宫颈癌细胞生长的研究

雷公藤甲素（triptolide,TPL）是传统中药雷公藤的主要活性成分,具有抗炎、抗肿瘤活性,但其毒副作用限制了临床上的广泛使用。为了探讨以TPGS-b-(PCL-ran-PGA)为载体制备的TPGS-b-(PCL-ran-PGA)/TPL纳米粒的表征和体外对宫颈癌细胞的抑制作用,采用乳化/溶剂挥发法,优化TPGS-b-(PCL-ran-PGA)与TPL比例,制备TPGS-b-(PCL-ran-PGA)/TPL纳米粒,对纳米粒进行表征,包括粒径大小、ζ电位、包封率、累积释放率,用MTS法体外研究游离型TPL和TPGS-b-(PCL-ran-PGA)/TPL纳米粒对宫颈癌细胞半数抑制浓度（IC50）,用克隆形成实验分析TPGS-b-(PCL-ran-PGA)/TPL纳米粒对宫颈癌细胞HeLa的抑制作用,用流式细胞仪分析纳米粒对HeLa细胞凋亡的影响。结果显示：当TPGS-b-(PCL-ran-PGA)与TPL为50∶1时制备的纳米粒粒径为(95.3±5.2)nm,zeta电位为(-12.2±0.9)mV,其累积释放曲线呈双相分布,TPGS-b-(PCL-ran-PGA)纳米粒对HeLa细胞在24、48和72 h的IC50（2.8、1.8、0.9 μg·L-1）远远低于游离型TPL（P<0.01）,克隆形成实验证明纳米粒能显著抑制肿瘤细胞生长,并能显著诱导HeLa细胞凋亡。研究结果表明,TPGS-b-(PCL-ran-PGA)/TPL纳米粒能抑制宫颈癌细胞HeLa的生长,其作用主要通过TPL和TPGS共同诱导细胞凋亡,可以作为抗宫颈癌等肿瘤的候选药物。     

Cloning and molecular modelling of turkey pancreatic lipase: structural explanation of the increased interaction power with lipidic interface

Starting from total pancreatic mRNAs, turkey pancreatic lipase (TPL) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the TPL is compared to that of human pancreatic lipase (HPL). A 3-D structure model of TPL was built using the 3-D structure of HPL as template, given the high amino acid sequence homology between the two lipases. Based on this model, the enhanced interaction power of TPL, as compared to that of HPL, into a phosphatidylcholine monolayer film, could be explained. We concluded that an increase in the exposed hydrophobic residues on the surface of TPL would be responsible for an enhanced interaction with a lipidic interface.     

Triptolide inhibits CD133+/CD44+ colon cancer stem cell growth and migration through triggering apoptosis and represses epithelial-mesenchymal transition via downregulating expressions of snail,slug, and twist

High recurrence and metastatic behavior patterns are the most important reasons for the failure of treatment strategies in patients with colon cancer. Cancer stem cells (CSCs), which are considered root of cancer, are thought to be associated with therapy resistance, relapse, and metastasis, and, therefore, targeting CSCs rather than the bulk population may be an effective approach. In cancer studies, there is an increasing interest in close friendship between epithelial-mesenchymal transition (EMT) and CSCs. Triptolide (TPL) isolated from Chinese herb Tripterygium wilfordii has important effects on the prevention of migration and metastasis as well as cytotoxic effect against cancer cells. The potential lethal efficacy of TPL on CSCs that is highly resistant to the drug is an unsolved mystery. Fundamentally, the present study basically aims to find answers to two questions: (a) is it possible to target colon CSCs with TPL? and (b) what are the mechanisms underlying TPL's potential to eliminate CSCs? Cytotoxic effects of TPL on CSCs were evaluated by WST-1 and Muse count and viability assays. Apoptosis assay and cell-cycle analysis were performed to investigate the inhibitory effect of TPL. Moreover, the effects of TPL on spheroid formation capacity, migration, and EMT processes, which are associated with CSC phenotype, were also investigated. The results revealed that TPL triggered cell death and apoptosis and altered cell cycle distribution. Moreover, TPL significantly reduced the snail slug and twist expressions associated with EMT. TPL has been shown to be effective in colon CSCs by in vitro experiments, and it might be a highly effective agent against colon cancer has been implicated in need of supporting in vivo and clinical studies.     

Cloning and expression of the Erwinia herbicola tyrosine phenol-lyase gene in Escherichia coli.

The tyrosine phenol-lyase (TPL) gene of Erwinia herbicola was cloned and expressed in Escherichia coli, and the complete nucleotide sequence of the gene determined. The TPL gene comprises 1368 bp, encoding 456 amino acids which have 90% amino acid identity with TPL from Citrobacter freundii. After replacing the 5'-flanking region of the TPL gene with the E. coli lac promoter, TPL protein could be hyperproduced constitutively in E. coli without induction by L-tyrosine.     

The nitroxide Tempol modulates anthracycline resistance in breast cancer cells

The occurrence of multidrug resistance (MDR) is the major obstacle to successful anthracycline-based cancer chemotherapy. In the present study, we assessed the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a piperidine nitroxide with growth-inhibitory properties in tumor cell lines, on a number of molecular mechanisms involved in the resistance of human breast adenocarcinoma cell lines to doxorubicin (DOX). Cytotoxicity studies in MCF-7 wildtype and their MDR variant MCF-7 Adr(R) cells showed a synergistic effect between TPL and DOX when exposure to TPL preceded or was simultaneous with DOX treatment in MCF-7 Adr(R) cells. This effect of TPL seems to be due in part to its ability to increase peroxide levels and to deplete cellular glutathione pools. In addition, TPL increased DOX accumulation in MCF-7 Adr(R) cells by interfering with P-glycoprotein-mediated DOX efflux, as evidenced using a specific antibody that recognizes the active form of the protein. TPL was also found to affect the expression levels of proteins involved in response to drug treatment (e.g., p53, bcl2, bax, p21). Taken together, our results indicate that TPL is a potential new agent that may improve the clinical effect of DOX in tumors exhibiting a MDR phenotype.     

Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase

Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate. These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates. We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations. Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E. coli Trpase, are essential for activity with both substrates. Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates. Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies. T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC. T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr. F448H TPL also has very low activity with L-Tyr. None of the mutant TPLs has any detectable activity with L-Trp as substrate. H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates. Thus, additional residues remote from the active site may be needed for substrate specificity. Both Trpase and TPL may react by a rare S(E)2-type mechanism.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Protection against radiation oxidative damage in mice by Triphala

Protection against whole body gamma-irradiation (WBI) of Swiss mice orally fed with Triphala (TPL), an Ayurvedic formulation, in terms of mortality of irradiated animals as well as DNA damage at cellular level has been investigated. It was found that radiation induced mortality was reduced by 60% in mice fed with TPL (1g/kg body weight/day) orally for 7 days prior to WBI at 7.5 Gy followed by post-irradiation feeding for 7 days. An increase in xanthine oxidoreductase activity and decrease in superoxide dismutase activity was observed in the intestine of mice exposed to WBI, which, however, reverted back to those levels of sham-irradiated controls, when animals were fed with TPL for 7 days prior to irradiation. These data have suggested the prevention of oxidative damage caused by whole body radiation exposure after feeding of animals with TPL. To further understand the mechanisms involved, the magnitude of DNA damage was studied by single cell gel electrophoresis (SCGE) in blood leukocytes and splenocytes obtained from either control animals or those fed with TPL for 7 days followed by irradiation. Compared to irradiated animals without administering TPL, the mean tail length was reduced about three-fold in blood leukocytes of animals fed with TPL prior to irradiation. Although, similar protection was observed in splenocytes of TPL fed animals, the magnitude of prevention of DNA damage was significantly higher than that observed in leukocytes. It has been concluded that TPL protected whole body irradiated mice and TPL induced protection was mediated through inhibition of oxidative damage in cells and organs. TPL seems to have potential to develop into a novel herbal radio-protector for practical applications.     

Kinetic study of 3,4-dihydroxyphenyl-L-alanine synthesis in Citrobacter freundii cells immobilized in carrageenan

A kinetic study was carried out of the enzymatic synthesis of 3,4-dihydroxyphenyl-L-alanine (DOPA) by the Citrobacter freundii 62 cells, possessing tyrosine-phenol-lyase (TPL) activity, immobilized in carrageenan, and optimum conditions of the reaction were found. The dependence of the TPL activity and its stability on the conditions of the DOPA synthesis was investigated. The TPL activity was higher and more stable in the immobilized cells as compared to free ones.     

Evaluation of the effects of dietary particle fractions on fermentation profile and concentration of microbiota in the rumen of dairy cows fed grass silage-based diets

The study evaluated the effects of three different theoretical particle lengths (TPL) of grass silage on the distribution of particle fractions of the diet and the resulting effects on fermentation profile and concentrations of protozoa and mixed bacterial mass in the rumen of three lactating Holstein cows fed total mixed rations (45% grass silage, 5% grass hay and 50% concentrate) ad libitum. Decreasing TPL of grass silage (long, medium, short) reduced particles retained on the 19-mm sieve of the Penn State Particle Separator, while particle fractions from 8 mm to 19 mm and smaller than 8 mm were increased. Different TPL did not affect pH and the concentration of volatile fatty acids in the rumen. However, lowering the TPL from long to medium increased significantly the bicarbonate concentration, acetate proportion and protozoal number in the rumen, whereas the proportion of bacterial protein in ruminal digesta and its amino acid concentration were significantly increased by the short TPL. For the current feeding conditions, it can be concluded that increasing the fraction of particles between 8 and 19 mm and probably even the fraction below 8 mm by decreasing TPL of grass silage do not adversely affect rumen conditions and can be beneficial in terms of optimising concentration and activity of ruminal microbiota in high-yielding dairy cows.     

Inhibition of tyrosine phenol-lyase from Citrobacter freundii by 2-azatyrosine and 3-azatyrosine.

The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined. 2-Aza-DL-tyrosine and 3-aza-DL-tyrosine were synthesized by standard methods of amino acid synthesis, while the L-isomers were prepared from 3-hydroxypyridine and 2-hydroxypyridine, respectively, with TPL (Watkins, E. B., and Phillips, R. S. (2001) Bioorg. Med. Chem. Lett. 11, 2099-2100). 3-Azatyrosine was examined as a potential transition state analogue inhibitor of TPL. Both compounds were found to be competitive inhibitors of TPL, with K(i) values of 3.4 mM and 135 microM for 3- and 2-aza-L-tyrosine, respectively. Thus, 3-azatyrosine does not act as a transition state analogue, possibly due to the lack of tetrahedral geometry at C-1. However, 2-aza-L-tyrosine is the most potent competitive inhibitor of TPL found to date. The K(i) value of 2-aza-L-tyrosine is half that of 2-aza-DL-tyrosine, indicating that the D-enantiomer is inactive as an inhibitor. Neither azatyrosine isomer was shown to be a substrate for beta-elimination, based on coupled assays with lactate dehydrogenase and on HPLC measurements. Both isomers of azatyrosine form equilibrium mixtures of external aldimine and quinonoid intermediates when they bind to TPL. However, 2-azatyrosine reacts about 10-fold faster to form a quinonoid intermediate than does 3-azatyrosine. Since 2-azatyrosine is in the zwitterion or phenolate ion form at all the pH values examined, the strong binding of this compound suggests that L-tyrosine may be bound to the active site of TPL as the phenolate anion.     

Polynitroxyl-albumin (PNA) enhances myocardial infarction therapeutic effect of tempol in rat hearts subjected to regional ischemia-reperfusion

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a low molecular weight stable nitroxyl radical (nitroxide), has been demonstrated in many in vitro and in vivo models to have protective effects against oxidative stress. The beneficial effect of TPL, however, is limited because of its short life-time in tissues. We have previously shown that polynitroxylated macromolecules such as polynitroxyl-human serum albumin (PNA) enable maintaining a sustained concentration of TPL for longer periods in tissues. PNA itself has previously been shown to inhibit ischemia-reperfusion (I/R) injury in the gut and to potentiate the activity of TPL. The aim of the present study was (i) to select an optimum formulation of PNA + TPL for therapeutic applications using in vivo EPR spectroscopy and (ii) to evaluate the efficacy of the PNA + TPL formulation in preventing I/R injury to heart, in an in vivo rat model. Rats were subjected to 45 min occlusion of the left anterior descending (LAD) coronary artery followed by 120 min reperfusion. PNA (100 mg/ml) + TPL (10 mg/ml), human serum albumin (HSA, 100 mg/ml) + TPL (10 mg/ml), or saline were injected 5 min before ischemia (3 ml/kg BW, i.v.) and 5 min before reperfusion (3 ml/kg BW, i.v.), followed by a 4 ml/kg BW infusion over 2 h reperfusion. Myocardial risk and infarct regions were then estimated. The results showed that the infarct volume, expressed as a percentage of the risk region, in the group treated with PNA + TPL was 39.7 +/- 3.1%, which was significantly smaller than for the saline (51.3 +/- 3.5%) or HSA + TPL (48.4 +/- 1.4%) groups. The results demonstrate that the PNA + TPL combination is very effective in reducing myocardial ischemia-reperfusion injury.     

Early Modern Humans and Morphological Variation in Southeast Asia: Fossil Evidence from Tam Pa Ling,Laos

Little is known about the timing of modern human emergence and occupation in Eastern Eurasia. However a rapid migration out of Africa into Southeast Asia by at least 60 ka is supported by archaeological, paleogenetic and paleoanthropological data. Recent discoveries in Laos, a modern human cranium (TPL1) from Tam Pa Ling‘s cave, provided the first evidence for the presence of early modern humans in mainland Southeast Asia by 63-46 ka. In the current study, a complete human mandible representing a second individual, TPL 2, is described using discrete traits and geometric morphometrics with an emphasis on determining its population affinity. The TPL2 mandible has a chin and other discrete traits consistent with early modern humans, but it retains a robust lateral corpus and internal corporal morphology typical of archaic humans across the Old World. The mosaic morphology of TPL2 and the fully modern human morphology of TPL1 suggest that a large range of morphological variation was present in early modern human populations residing in the eastern Eurasia by MIS 3.     

The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+). Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation. Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination. Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+). Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process. In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+). E69D TPL shows no change in fluorescence under these conditions. High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL. This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.     

Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.