• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Copper interactions with DNA of chromatin and its role in neurodegenerative disorders

    2013-12-23 06:15:18GovinrjuShkrStshVsuvRjuSmsivRoRoRjmm
    Journal of Pharmaceutical Analysis 2013年5期

    M. Govinrju, H.S. Shkr, S.B. Stsh, P. Vsuv Rju,K.R. Smsiv Ro, K.S.J. Ro, A.J. Rjmm

    aMolecular Biophysics Unit, Indian Institute of Science, Bangalore, India

    bDepartment of Pharmacy Practice, KIMS Hospital and Research Center, VIPS, Bangalore, India

    cDepartment of Pharmaceutics, Acharya & B.M. Reddy College of Pharmacy, Bangalore 560107, India

    dDepartment of Neuroscience, Medical University of South Carolina, Charlton, USA

    eDepartment of Biotechnology, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, India

    fCentre for Neuroscience, Institute for Scientific Research and Technological services, INDICASAT-AIP, City of Knowledge,Republic Panama, Germany

    gDepartment of Pharmacognosy, KLEU's College of Pharmacy, Bangalore 560010, India

    1. Introduction

    Chromatin is the combination of DNA and proteins that make up the contents of the nucleus of eukaryotic cells. The primary functions of chromatin are:to package DNA into a smaller volume to fit in the cell, to strengthen DNA to allow mitosis and meiosis and prevent DNA damage.It also helps to control gene expression and DNA replication [1]. An alteration in the chromatin organization may lead to the neuronal cell death and loss of regulation of DNA methylation, leading to altered gene expression as observed in many neurodegenerative disorders [2].

    The chromatin structure is susceptible to change under conditions of ionic strength,pH,temperature and interaction of divalent metal ions with DNA [3]. The interaction of copper ions with DNA has been of particular interest because of the involvement of copper ions in regular activities such as cellular respiration and neurotransmitter biosynthesis etc.

    Copper works as cofactor for numerous enzymes and plays an important role in the development of central nervous system.However, excessive levels or perturbation of copper metabolism can lead to accumulation of copper preferentially in heterochromatin regions and cause intracellular toxicity [4,5]. The redox properties of copper can cause oxidative damage to DNA. Copper induced DNA damage may probably lead to neuronal dysfunction, critical failure of biological functions and ultimately cell death contribute to neurological disease [6]. Copper functions as a “double whammy”in the brain by generating large number of DNA attacking reactive oxygen species (ROS) via a Fenton reaction, which causes catastrophic damage to lipids, proteins and DNA [7].

    Copper can also directly bind to protein and DNA leading to structural and functional modifications and is involved in chromatin condensation. For this reason, copper is one of the strongly suspected etiological factors in neurodegenerative disorders such as, Alzheimer's disease, Parkinson's disease, Huntington disease and familial amyotrophic lateral sclerosis [8].

    Here we have studied the properties of chromatin with respect to conformational changes and damage as a consequence of interaction with copper.Structural and conformational aspects of the interaction of copper with chromatin are studied by a variety of spectroscopic techniques including UV-visible spectrophotometry, optical melting studies, circular dichroism (CD), circular dichroism melting studies and fluorescence spectroscopy. Temperature studies provide the basis to understand the factors that dictate the stability and structure of chromatin in the presence of copper.

    2. Materials and methods

    2.1. Reagents

    Copper chloride dihydrate, CuCl2·2H20 (Merck Schuchard), ethidium bromide (EB), C21H20BrN3, (Amersham Life Sciences) and Tris buffer (Sigma), were purchased and used without further purification.

    The stock solutions of copper chloride (50 mM) and EB (5 mg/mL)were prepared using Milli-Q water and stored at 4°C in the dark until use.The stock solution of Tris buffer was prepared by dissolving 2.4 g of Tris in 200 mL of Milli-Q water. The pH of the buffer was set by the addition of dilute HCl and measured using digital pH meter with a combined glass electrode (EUTECH Instruments).

    2.2. Isolation of the nuclei from the brain samples

    Nuclei were isolated from the cortex region of the human brain(Brain bank, JSS Medical College, Mysore) according to the method described by Usha Rani et al., 1986 [9]. In brief, brain tissue was weighed (5 g) and perfused with normal saline to remove any blood in the tissue. Brain tissue was cut into small pieces and minced thoroughly. The minced brain tissue was homogenized in 0.34 M sucrose in buffer-A (50 mM Tris—HCl pH 7.5, 25 mM KCl, 5 mM MgCl2, and 0.5 mM PMSF) using homogenizer. The homogenate was filtered through 2 layers of cheese cloth and the filtrate was centrifuged at 1000 g (3500 rpm)for 10 min at 4°C.The supernatant was decanted carefully and the pellet was resuspended in 1 M sucrose in buffer-A.The suspended pellet was homogenized using hand held homogenizer and centrifuged at 100,000 g (42,000 rpm) in ultracentrifuge for 1 h.The pellet obtained was washed with 1 M sucrose in buffer-A,0.34 M sucrose and 0.34 M sucrose with 0.1% Triton X-100,respectively.The pellet obtained was dissolved in Tris—HCl buffer and the concentration of the nuclear suspension was determined by taking absorbance at A260in 1 mL of 2 M NaCl or 5 M urea.

    2.3. Preparation of nuclei and soluble chromatin

    Soluble chromatin was prepared from the isolated nuclei by limited digestion of nuclei with micrococcal nuclease [10].Nuclear suspension (100 μL) was mixed with 0.1 M CaCl2and incubated at 37°C for 2 min. After incubation period, the nuclear suspension was digested with micrococcal nuclease (50 units) by incubating at 37°C for 1 min.The reaction was stopped by 0.25 M EDTA and centrifuged at 5000 rpm for 5 min.The pellet obtained was suspended in 10 mM NaHSO3and 1 mM EDTA (pH 7.5).The soluble chromatin was used for the copper interaction studies.

    2.4. Copper-chromatin binding studies

    2.4.1. Spectrophotometric method

    Spectrophotometric binding studies of copper with chromatin were performed to understand the nature of the conformational changes of chromatin on binding with copper.The electronic absorption studies were investigated at pH 7.4 using a Jasco V-530 Spectrophotometer equipped with a Peltier temperature controller. Chromatin sample was prepared in Tris—HCl buffer (5 mM, pH 7.4, 400 μL) in the presence and absence of CuCl2(50 μM and 100 μM). Absorbance spectrum was measured at wavelength between 210 nm and 320 nm with a matched set of 1 cm path length quartz cuvettes. Buffer baseline was subtracted with the Jasco software and the resultant spectrum was recorded.

    2.4.2. Circular dichroism studies

    Circular dichroism (CD) spectroscopy is one of the most sensitive techniques available for monitoring conformational properties of DNA in solution. Copper induced conformational change of chromatin was measured on a Jasco J-715 Spectro polarimeter at 25°C. Spectra were recorded using a path length of 1 mm quartz cuvette at 1 nm intervals in the wavelength between 200 and 320 nm. An average of four repetitive scans using a scan speed of 20 nm/min was taken into consideration. Chromatin sample was prepared in Tris—HCl buffer(5 mM,300 μL,pH 7.4)in the absence and presence of different concentrations of CuCl2(50, 100 and 500 μM). Buffer background was subtracted by using the built-in feature of Jasco software and the resultant spectrum was recorded.

    2.5. Thermal denaturation studies

    2.5.1. Spectrophotometric method

    UV-thermal denaturation of chromatin in the presence and absence of CuCl2was measured with Jasco V-530 spectrophotometer equipped with Jasco ETC-505T temperature controller and cell holder that permits temperature control using the temperature control program.

    2.5.2. Circular dichroism melting studies

    Melting studies of chromatin in the presence and absence of CuCl2were performed on a Jasco J-715 spectropolarimeter connected to model PTC-348WI, peltier type temperature control system.Samples were recorded at wavelengths between 200 nm and 320 nm by varying the temperature from 20oC to 100oC.

    2.6. Fluorescence studies of DNA structure and dynamics

    EB replacement experiment was carried out to verify the local structural information due to the interaction of copper with chromatin. Fluorescence emission studies were carried out using equimolar concentrations (1:1) of chromatin and EB. The EB binding pattern of chromatin and the effect of different concentrations of CuCl2(100—500 μM) on the EB fluorescence were analyzed. Samples of DNA/EB solutions were excited at 530 nm and emission spectra were recorded from 550 nm to 650 nm using Jasco J-600 spectrofluorimeter.

    3. Results

    3.1. Copper-chromatin binding studies

    Spectrophotometric binding studies help in understanding ability of chromatin to bind to copper and the nature of conformational changes that occur to chromatin upon binding with copper. The absorbance spectra of chromatin show the absorption maximum at 265 nm as shown in Fig.1A.Upon addition of CuCl2(50 μM and 100 μM),there was an increase in the absorbance with blue shift to 260 nm.On further addition of copper, no change in the intensity was noticed indicating saturation.

    Circular dichroism (CD) is a useful technique for studying conformational changes and the degree of asymmetry of bases of DNA in chromatin [11]. Structural transition of chromatin in the absence of copper, exhibits a characteristic positive peak at 275 nm(Fig.2A). CD spectra of chromatin between 250 and 300 nm are dominated by DNA and proteins contribute very little to CD spectra of this region[12—14].Upon addition of copper to chromatin,a decrease in the magnitude of both the positive and the negative bands was noticed.This is due to complex formation between Cu2+and the bases of DNA. This was accompanied by a conformational change with a cross over point at 245 nm as illustrated in Fig.2A.

    3.2. Thermal denaturation studies of chromatin

    The thermal behavior of chromatin was monitored using UV/vis absorbance spectroscopy. The melting temperature curves of DNA are enhanced as the temperature increases and stabilized the DNA in chromatin as shown in Fig.1B and C. Identical melting curves are produced free chromatin and chromatin with 50 μM copper.Melting profile of chromatin shown in Fig.1D reveals two transitions, the first transition at 58.8°C and the second at 70.27°C.Upon addition of 100 μM copper, Tm shifts to 60°C and 72.5°C.

    Fig.1 (A)Absorbance spectra of chromatin with varied concentrations of copper.(B)Absorbance spectra of free chromatin at varied temperature.(C)Absorbance spectra of chromatin with 50 μM copper at varied temperature. (D) Melting profile of free chromatin and chromatin with 100 μM copper.

    Fig.2 (A) Circular dichroism studies of chromatin with varied concentrations of copper. (B) Circular dichroism studies of chromatin at varied temperature. (C) Circular dichroism studies of chromatin with 50 μM copper at varied temperature. (D) Circular dichroism studies of chromatin with 100 μM copper at varied temperature. (E) Melting profile of free chromatin and chromatin with 500 μM copper at 275 nm wavelength.

    Thermal denaturation studies of chromatin in the absence of copper at variable temperature from 20°C to 105°C are shown in Fig.2B. Effect of 50 μM copper is shown in Fig.2C, and 100 μM copper is shown in Fig.2D, the melting profile of chromatin is also shown in Fig.2E. There is an increase in intensity of both positive and negative bands at the isodichroic point at 227 nm for chromatin and at 229 nm in the presence of copper.These results indicated that the CD transition happens in two states and the structural change was cooperative. It also confirms identical melting profiles of native chromatin and chromatin with 50 μM and 100 μM copper [15].The CD melting profile in the presence and absence of copper at 275 nm shown in Fig.2E is evidence for the above results.

    3.3. Fluorescence studies of DNA structure and dynamics

    The fluorescence spectra of chromatin—EB complex excited at 530 nm and emission spectra were scanned from 550 to 650 nm as shown in Fig.3A. Upon addition of copper to chromatin, the fluorescence intensity of chromatin—EB complex is decreased with increasing concentration of copper.

    4. Discussion

    4.1. Copper-chromatin binding studies

    The absorbance spectra of chromatin in this wavelength region(Far UV)of the absorption spectra are sensitive to π—π*transitions of the electrons of the purine and pyrimidine rings. This is due to the increased positive base pair tilting of conformational change in DNA [16]. The hyperchroism and blue shift (hypsochromic shift)is due to binding of copper to the bases of DNA in chromatin.Hydrogen bonds are disrupted by the process of partial unwinding which induces structural changes such as loosening of base—base interaction, base tilting and destabilization of the DNA double helix leading to DNA denaturation [17]. The destabilization was due to most probable binding sites in DNA such as Cu2+to N7of guanine and N3of cytosine in line with our present results and with the predictions of Eichhorn and Clark [18,19].

    CD spectra of chromatin showed a characteristic positive peak at 275 nm (Fig.2A) due to base stacking and a negative peak at 210 nm due to protein content. CD spectra of chromatin between 250 and 300 nm are dominated by DNA, chromatin as proteins contribute very little to CD spectra of this region. Upon gradual addition of copper to chromatin, a decrease in the magnitude of both the positive and negative bands was noticed. These data indicate that cationic copper binds to the anionic phosphate ions of the DNA back bone,consequently,the lengthening of DNA which induced the loss of conformation. Copper first binds to anionic phosphate [20] groups of the backbone and subsequently to the bases, specifically to guanine [21] and cytosine [22] of DNA by competing with hydrogen bands. This disrupts the interactions between the bases of DNA and thereby weakening base stacking

    Fig.3 Fluorescence emission spectra of free chromatin and chromatin with copper.

    [23] by tilting the bases leads to change in the winding angle.As the winding angle increases, the magnitude of the band decreases with a greater degree of twisting of the chain.Hydrogen bonding forces play an essential role in the binding [24,25] which may affect sugar puckering and change in the conformation of guanine from anti to syn [19]. The negative super helical tension and rearrangement in coordination caused by copper may drive local transitions to alternate conformational change in the DNA structures in chromatin.Binding of Cu2+with adenine is similar in interaction with the N7 position and the phosphate site would represent a third copper-base binding site.Copper does not bind to thymine but both the bases of GC pair are known to involve in the copper complexes. DNA conformation is an important aspect for the gene expression. This study provides evidence of copper induced DNA damage in the chromatin organization and neuronal cell death, which is implicated in many neurological disorders.

    4.2. Thermal denaturation studies

    This study provides information about binding affinity of copper with DNA in chromatin and subsequent conformational changes to DNA.It is known that double stranded DNA gradually dissociates to single strands with increasing temperature [26]. Tm is strictly related to the stability of the double helix and the interaction of copper with DNA.

    The thermal denaturation experiments by both UV and CD are represented by two transitions. The small variation in Tm is evidence that copper binds to anionic phosphate backbone and to the bases depending upon the accessibility of DNA in chromatin.The results of CD melting studies show that protein bound to DNA in chromatin stabilizes DNA. Higher stabilization is due to the compact binding of histones. Both CD and UV melting results indicate that there is not much effect of temperature on DNA in chromatin. This may be because the DNA bases in chromatin are protected by associating with the chromatin proteins, so that the bases are accessible for copper [27]. It is concluded that the basic conformation of DNA in native chromatin is determined largely by histones and nonhistone proteins. It is also seen that DNA is greatly stabilized against thermal melting in the DNA—histone complex. The small increase in Tm indicates that copper interacts with DNA in chromatin and changes the conformation of chromatin structure. It is also evidence that DNA is greatly stabilized against thermal melting in the DNA—histone complex.

    4.3. Fluorescence studies of DNA structure and dynamics

    Fluorescence spectroscopy is an important technique for probing the structure and dynamics of nucleic acids. The utility of fluorescence techniques stems the ability of fluorophores to reflect changes in their molecular environment through measurable alterations in emission properties. The decrease in chromatin—EB emission with the addition of copper indicates that binding of Cu2+ions with chromatin —EB complex forms a new nonfluorescent complex of Cu—chromatin—EB, which causes the fluorescence quenching of chromatin—EB complex [28]. These data show that Cu2+ions bind to DNA in chromatin, resulting in chemical DNA denaturation and the binding of copper is mainly concentration dependent.

    5. Conclusion

    Our study showed that copper binds to bases of DNA in chromatin by changing the winding angle of the helix. This induces the DNA damage and alters the B-conformation. Altered B-DNA conformation will alter the integrity of DNA which may affect the normal process of DNA replication and transcription.Copper induced DNA damage in the brain may cause neurotoxicity and the neuronal cell death and is implicated in Alzheimer's disease and other neurological disorders.Hence copper is expected to become one of the key factors for causing neurodegeneration. It is also concluded that histone and nonhistone proteins present in chromatin protect DNA from oxidative DNA damage and slowdown the age related diseases.

    We thank the Chairman, Prof. S.P. Siddhartha, Molecular Biophysics Unit,Indian Institute of Science,Bangalore,for helpful discussions and valuable comments of this research and suggestions.The authors of this publication wish to thank Government of India for financial support to Molecular Biophysics Unit, Indian Institute of Science, Bangalore.

    [1] R.D. Kornberg, Structure of chromatin, Annu. Rev. Biochem. 46(1977) 931—954.

    [2] R.D. Kornberg, Chromatin structure: a repeating unit of histones and DNA, Science 184 (1974) 868—871.

    [3] J.L.Sagripanti,P.L.Goering,A.Lamanna,Interaction of copper with DNA and antagonism by other metals,Toxicol.Appl.Pharmacol.110(1991) 477—485.

    [4] S. Scarpa, A.D. Fuso, F. Anselmi, et al., Presenilin 1 gene silencing by S-adenosylmethionine: a treatment for Alzheimer disease, FEBS Lett. 541 (2003) 145—148.

    [5] B. Halliwell, J.M.C. Gutteridge, Oxygen toxicity, oxygen radicals,transition metals and disease, Biochem. J. 219 (1984) 1—14.

    [6] D. Strausak, J.F. Mercer, H.H. Dieter, et al., Multhaup, Copper in disorders with neurological symptoms: Alzheimer's, Menkes, and Wilson diseases, Brain Res. Bull. 55 (2001) 175—185.

    [7] D.E.Hartter,A.Barnea,Brain tissue accumulates copper by two liganddependent saturable processes, J. Biol. Chem. 263 (1988) 799—805.

    [8] T.A. Rouault, Systemic iron metabolism: a review and implications for brain iron metabolism, Pediatr. Neurol. 2 (2001) 130—137.

    [9] B. Usha Rani, K.S. Rao, DNA and DNase in isolated neuronal,astrocyte and oligodendrocyte cell enriched fraction from young and old chick brain, Indian J. Biochem. Biophys. 23 (1986) 279—282.

    [10] J.R.Korenberg,S.M.Pulst,R.L.Neve,et al.,The Alzheimer amyloid precursor protein maps to human chromosome 21 bands q21.105-q21.05, Genomics 5 (1) (1989) 124—127.

    [11] P. Vasudevaraju, T. Bharathi, Jyothsna et al. New evidence on iron,copper accumulation and zinc depletion and its correlation with DNA integrity in aging human brain regions, Indian J. Psychiatry 52 (2)(2010) 140—144.

    [12] A.J. Adler, G.D. Fasman, L.J. Wangh, et al., Altered conformational effects of naturally acetylated Histone f2a1(IV) in f2al-DNA complexes, J. Biol. Chem. 249 (1974) 2911—2914.

    [13] I. Sissoeff, J. Grisvard, E. Guille, Studies on metal ions-DNA interactions: specific behaviour of reiterative DNA sequences, Prog.Biophys. Mol. Biol. 31 (1976) 165—199.

    [14] F.E.Rosetto,E.Nieboer,The Interaction of metal ions with synthetic DNA:Induction of conformational and structural transitions,J.Inorg.Biochem. 54 (1994) 167—186.

    [15] J. Dugoid, V.A Bloomfield, J. Benevides, Raman spectroscopy of DNA—metal complexes.I.Interactions and conformational effects of the divalent cations: Mg,Ca,Sr,Mn,Co,Ni,Cu,Pd and Cd, Biophys. J.65 (1993) 1916—1928.

    [16] J.L Mergny, Li Jing, Lacroix Laurent, et al., Thermal difference spectra:a specific signature for nucleic acid structures,Nucleic. acids Res. 33 (16) (2005) e138.

    [17] C.Zimmer,G.Luck,H.Fritzsche,DNA—copper(II)complex and the DNA conformation, Biopolymers 10 (1971) 441—463.

    [18] G.L. Eichhorn, P. Clark, Interactions of metal ions with polynucleotides and related compounds. v. the unwinding and rewinding of DNA strands under the influence of copper(II)ions,Proc.Natl.Acad.Sci. USA 53 (1965) 586—593.

    [19] P. Clark, G.L. Eichhorn, A Simple probe for DNA accessibility in chromatin, J. Inorg. Biochem. 19 (1995) 765—772.

    [20] H. Fritzsche, C. Zimmer, IR studies of DNAs, their constituents and analogues.4.The binding sites of Cu (II) in DNA, Eur. J. Biochem. 5(1968) 42—44.

    [21] M.N.Dehkord,A.K.Bordbar,P.Lincoln,Spectroscopic study on the interaction of ct-DNA with manganese salen complex containing triphenyl phosphonium groups, Spectrochim. Acta A Mol. Biomol.Spectrosc. 90 (2012) 50—54.

    [22] W. Forster, E. Bauer, H. Schut, Thermodynamics and kinetics of the interaction of copper (II)ions with Native DNA, Biopolymers 18(1979) 625—661.

    [23] Y. Courtois, P. Fromageot, W. Guschlbaue, Protonated Polynucleotide Structures. 3. An Optical Rotatory Dispersion Study of the Protonation of DNA, Eur. J. Biochem. 56 (4) (1968) 493—501.

    [24] G. Goutam, B. Manju, V. Sashishekaran, Conformational flexibility of DNA: Polymorphism and handedness,Proc. Natl.Acad.Sci.USA 77 (11) (1980) 6486—6490.

    [25] G.L. Eichhorn, P. Clark, Interactions of metal ions with polynucleotides and related compounds v. The unwinding and rewinding of DNA strands under the influence of copper(II)ions,Proc.Nat.Acad.Sci. USA 53 (1965) 586—593.

    [26] R. Mandel, G.D. Fasman, Thermal denaturation of DNA and polypeptide complexes. Simultaneous absorption and circular dichroism measurements,Biochem.Biophys.Res.Commun.59(20)(1974)672—679.

    [27] C. Zimmer, G. Luck, H. Triebel, Conformation and reactivity of DNA, 4. Base binding ability of transition metal ions to native DNA and the effect of helix conformation with special reference to the DNA- Zn (II) complex, Biopolymers 13 (3) (1974) 425—454.

    [28] John Olmsted 111, R.K. David, Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids, Biochemistry 16 (16) (1977) 3647—3654.

    国产熟女欧美一区二区| 亚洲国产精品999| 三级国产精品片| 亚洲,一卡二卡三卡| 成人国语在线视频| 亚洲人成网站在线观看播放| 国产日韩欧美亚洲二区| 欧美日韩亚洲高清精品| 热re99久久国产66热| 免费av中文字幕在线| 亚洲国产av新网站| 美女大奶头黄色视频| 久久久久久久久久久免费av| 免费看不卡的av| 亚洲精品在线美女| 欧美国产精品va在线观看不卡| 欧美日韩视频高清一区二区三区二| 免费播放大片免费观看视频在线观看| 久久精品亚洲av国产电影网| 久久国产精品大桥未久av| 新久久久久国产一级毛片| 女人被躁到高潮嗷嗷叫费观| 国产精品嫩草影院av在线观看| 制服人妻中文乱码| 亚洲综合色惰| 一级毛片 在线播放| 亚洲欧美一区二区三区久久| 免费不卡的大黄色大毛片视频在线观看| 丝袜美腿诱惑在线| 久久精品国产a三级三级三级| 激情视频va一区二区三区| 亚洲国产欧美网| www.熟女人妻精品国产| www.精华液| 大码成人一级视频| 日产精品乱码卡一卡2卡三| 欧美精品高潮呻吟av久久| 国产日韩欧美亚洲二区| 亚洲国产成人一精品久久久| 亚洲五月色婷婷综合| 亚洲精品日本国产第一区| 春色校园在线视频观看| av卡一久久| av在线播放精品| 国产高清国产精品国产三级| 免费av中文字幕在线| 国产精品亚洲av一区麻豆 | 欧美日韩精品网址| 丰满少妇做爰视频| 好男人视频免费观看在线| 天美传媒精品一区二区| 欧美中文综合在线视频| 精品一区二区免费观看| 黄片无遮挡物在线观看| 男女啪啪激烈高潮av片| 91国产中文字幕| 中文天堂在线官网| 久久免费观看电影| 在线天堂最新版资源| videosex国产| 日本欧美视频一区| 熟妇人妻不卡中文字幕| 黄色 视频免费看| 男人爽女人下面视频在线观看| 国产精品久久久久久av不卡| 亚洲av电影在线观看一区二区三区| 日韩三级伦理在线观看| 国产熟女午夜一区二区三区| 伊人亚洲综合成人网| 国产成人精品久久二区二区91 | 亚洲婷婷狠狠爱综合网| 天美传媒精品一区二区| 国产亚洲最大av| 日韩精品有码人妻一区| 91国产中文字幕| 亚洲天堂av无毛| a级毛片在线看网站| 亚洲一码二码三码区别大吗| 婷婷色综合大香蕉| 黄色怎么调成土黄色| 亚洲 欧美一区二区三区| 校园人妻丝袜中文字幕| 亚洲色图综合在线观看| 亚洲,一卡二卡三卡| 午夜福利视频精品| 91精品国产国语对白视频| 日日爽夜夜爽网站| av国产久精品久网站免费入址| 黄片播放在线免费| 9色porny在线观看| 久久影院123| 久久久久久久久免费视频了| 久久精品久久精品一区二区三区| 人人妻人人添人人爽欧美一区卜| 国产片内射在线| 国产人伦9x9x在线观看 | 亚洲精品中文字幕在线视频| 一边摸一边做爽爽视频免费| 婷婷色av中文字幕| 久久久久久人妻| 新久久久久国产一级毛片| 国产精品麻豆人妻色哟哟久久| 久久久久视频综合| 午夜激情久久久久久久| 久久久久人妻精品一区果冻| 久久97久久精品| 嫩草影院入口| 在线观看美女被高潮喷水网站| 天堂8中文在线网| 久久韩国三级中文字幕| 看免费av毛片| 亚洲婷婷狠狠爱综合网| 丰满少妇做爰视频| 人妻少妇偷人精品九色| av电影中文网址| 一本大道久久a久久精品| 亚洲精品国产av成人精品| 中文字幕精品免费在线观看视频| 春色校园在线视频观看| 中文字幕精品免费在线观看视频| 色婷婷久久久亚洲欧美| 欧美人与性动交α欧美软件| 夜夜骑夜夜射夜夜干| 老司机亚洲免费影院| 天天躁日日躁夜夜躁夜夜| 一区二区三区乱码不卡18| 亚洲成人手机| 男男h啪啪无遮挡| 丰满饥渴人妻一区二区三| 999久久久国产精品视频| 国产精品不卡视频一区二区| 国产福利在线免费观看视频| 99久久中文字幕三级久久日本| 丝袜美足系列| 日韩 亚洲 欧美在线| 男人舔女人的私密视频| 成年女人在线观看亚洲视频| 不卡视频在线观看欧美| av有码第一页| 亚洲精品日韩在线中文字幕| 99热网站在线观看| av在线观看视频网站免费| 国产国语露脸激情在线看| 一边亲一边摸免费视频| 国产综合精华液| av卡一久久| 不卡av一区二区三区| 在现免费观看毛片| av女优亚洲男人天堂| 中文乱码字字幕精品一区二区三区| 99久久精品国产国产毛片| 一本大道久久a久久精品| 久久青草综合色| 大码成人一级视频| 1024视频免费在线观看| 一级,二级,三级黄色视频| 精品人妻偷拍中文字幕| 日韩av在线免费看完整版不卡| 国产精品 国内视频| 寂寞人妻少妇视频99o| 国产黄色免费在线视频| 中文欧美无线码| 久久久精品国产亚洲av高清涩受| 99香蕉大伊视频| 有码 亚洲区| 啦啦啦中文免费视频观看日本| 国产亚洲欧美精品永久| 亚洲欧洲精品一区二区精品久久久 | 亚洲色图 男人天堂 中文字幕| 久久久久久伊人网av| 伦理电影免费视频| 一区二区三区四区激情视频| 最近手机中文字幕大全| 亚洲欧美一区二区三区国产| 亚洲精品中文字幕在线视频| 国产又爽黄色视频| 国产探花极品一区二区| 欧美激情高清一区二区三区 | 免费观看av网站的网址| 亚洲精品自拍成人| 久久久久久久久久久久大奶| 18在线观看网站| 国产成人91sexporn| √禁漫天堂资源中文www| 在线观看免费高清a一片| 中文字幕精品免费在线观看视频| 一本久久精品| 老司机亚洲免费影院| 秋霞在线观看毛片| 老汉色∧v一级毛片| 国产亚洲精品第一综合不卡| 精品一品国产午夜福利视频| 人人妻人人爽人人添夜夜欢视频| 女性生殖器流出的白浆| 91午夜精品亚洲一区二区三区| 亚洲国产av影院在线观看| 美女xxoo啪啪120秒动态图| 中文字幕精品免费在线观看视频| 香蕉精品网在线| 成年动漫av网址| 午夜福利在线免费观看网站| 美女主播在线视频| 夫妻午夜视频| 日韩欧美精品免费久久| 丝袜在线中文字幕| 久久久精品94久久精品| 大片免费播放器 马上看| 水蜜桃什么品种好| 香蕉精品网在线| 免费看不卡的av| 边亲边吃奶的免费视频| 男女国产视频网站| 性色av一级| 欧美激情极品国产一区二区三区| 婷婷色综合大香蕉| 毛片一级片免费看久久久久| 欧美精品亚洲一区二区| 国产爽快片一区二区三区| 亚洲欧美色中文字幕在线| 黄色配什么色好看| 婷婷色麻豆天堂久久| 少妇的丰满在线观看| 久久ye,这里只有精品| 91精品伊人久久大香线蕉| 老司机影院成人| 秋霞在线观看毛片| 九九爱精品视频在线观看| 赤兔流量卡办理| 国产白丝娇喘喷水9色精品| 大香蕉久久成人网| 久久久精品94久久精品| 又黄又粗又硬又大视频| 中文字幕制服av| 久久这里有精品视频免费| 人人妻人人爽人人添夜夜欢视频| 亚洲av日韩在线播放| 69精品国产乱码久久久| 91久久精品国产一区二区三区| 国语对白做爰xxxⅹ性视频网站| 老司机亚洲免费影院| 亚洲国产精品国产精品| 亚洲国产精品999| 2022亚洲国产成人精品| 久久午夜综合久久蜜桃| 亚洲 欧美一区二区三区| 又黄又粗又硬又大视频| 韩国高清视频一区二区三区| 国产深夜福利视频在线观看| 久久国产精品男人的天堂亚洲| 亚洲第一青青草原| 亚洲成色77777| 母亲3免费完整高清在线观看 | 七月丁香在线播放| 在线观看美女被高潮喷水网站| 最近的中文字幕免费完整| 在线观看人妻少妇| 女人久久www免费人成看片| 超色免费av| 熟女少妇亚洲综合色aaa.| 国语对白做爰xxxⅹ性视频网站| 久久久久久久国产电影| 99国产精品免费福利视频| a级毛片在线看网站| 啦啦啦在线观看免费高清www| av国产久精品久网站免费入址| 777久久人妻少妇嫩草av网站| 国产日韩欧美在线精品| 十八禁网站网址无遮挡| 只有这里有精品99| 一级片'在线观看视频| 一区二区三区乱码不卡18| 飞空精品影院首页| 国产探花极品一区二区| 亚洲国产av新网站| 成人18禁高潮啪啪吃奶动态图| 咕卡用的链子| 久久久精品区二区三区| h视频一区二区三区| 久久精品人人爽人人爽视色| 国产精品国产三级专区第一集| 一本—道久久a久久精品蜜桃钙片| 天堂8中文在线网| 国产成人aa在线观看| 久久久欧美国产精品| 大香蕉久久成人网| 中文精品一卡2卡3卡4更新| 国语对白做爰xxxⅹ性视频网站| 午夜精品国产一区二区电影| 女性生殖器流出的白浆| 最黄视频免费看| 亚洲精品av麻豆狂野| 中文字幕亚洲精品专区| 成年人免费黄色播放视频| 男人操女人黄网站| 国产综合精华液| 免费黄色在线免费观看| 天天影视国产精品| 久久女婷五月综合色啪小说| 制服诱惑二区| 成人漫画全彩无遮挡| 久久精品国产综合久久久| 丝袜在线中文字幕| 精品视频人人做人人爽| 中文字幕另类日韩欧美亚洲嫩草| 国产97色在线日韩免费| 精品国产国语对白av| videos熟女内射| 国产成人精品久久二区二区91 | 在线观看免费视频网站a站| 熟女电影av网| 欧美bdsm另类| 亚洲情色 制服丝袜| 少妇人妻精品综合一区二区| 国产xxxxx性猛交| 美女脱内裤让男人舔精品视频| 欧美日韩视频精品一区| 中文字幕最新亚洲高清| 男人爽女人下面视频在线观看| av在线app专区| 久久精品人人爽人人爽视色| 制服诱惑二区| 欧美精品av麻豆av| 男人爽女人下面视频在线观看| 亚洲精品一区蜜桃| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 一级片免费观看大全| 自拍欧美九色日韩亚洲蝌蚪91| 欧美 亚洲 国产 日韩一| 男女无遮挡免费网站观看| 亚洲一区二区三区欧美精品| 久久午夜综合久久蜜桃| 免费黄色在线免费观看| 另类亚洲欧美激情| 亚洲精品久久久久久婷婷小说| 国产高清国产精品国产三级| 免费黄色在线免费观看| 亚洲第一青青草原| 亚洲精品,欧美精品| 亚洲av电影在线观看一区二区三区| 亚洲一区中文字幕在线| 欧美av亚洲av综合av国产av | 久久久久国产网址| 日本wwww免费看| 一级毛片我不卡| 高清在线视频一区二区三区| 天堂中文最新版在线下载| 一本色道久久久久久精品综合| 欧美国产精品va在线观看不卡| 亚洲国产毛片av蜜桃av| 电影成人av| 在线观看国产h片| 国产精品免费视频内射| 久热这里只有精品99| 日本色播在线视频| 久久99热这里只频精品6学生| 国产精品蜜桃在线观看| 精品少妇黑人巨大在线播放| 久久精品久久久久久噜噜老黄| 丰满少妇做爰视频| 欧美av亚洲av综合av国产av | 考比视频在线观看| 国产女主播在线喷水免费视频网站| 国产一区亚洲一区在线观看| 亚洲av免费高清在线观看| 日本91视频免费播放| 中文字幕人妻丝袜制服| 最近最新中文字幕免费大全7| 性色avwww在线观看| 91成人精品电影| 99精国产麻豆久久婷婷| 久久久久国产一级毛片高清牌| 老司机影院成人| 亚洲视频免费观看视频| 日本色播在线视频| 王馨瑶露胸无遮挡在线观看| 国产免费又黄又爽又色| 母亲3免费完整高清在线观看 | 精品国产国语对白av| 精品亚洲成a人片在线观看| 精品少妇黑人巨大在线播放| 国产精品秋霞免费鲁丝片| 欧美日韩视频高清一区二区三区二| 中文字幕av电影在线播放| 日本爱情动作片www.在线观看| 狂野欧美激情性bbbbbb| 久久亚洲国产成人精品v| 成人黄色视频免费在线看| 人妻人人澡人人爽人人| 久久婷婷青草| 日本wwww免费看| 一本久久精品| 国产av一区二区精品久久| 久久精品亚洲av国产电影网| 久久精品久久精品一区二区三区| 天天躁夜夜躁狠狠躁躁| 国产男女超爽视频在线观看| 国产成人精品婷婷| 久久久精品区二区三区| 看十八女毛片水多多多| 国产男人的电影天堂91| 在线免费观看不下载黄p国产| 亚洲中文av在线| 国产一区二区在线观看av| 亚洲欧美中文字幕日韩二区| 亚洲精品美女久久久久99蜜臀 | 丝袜脚勾引网站| 岛国毛片在线播放| 欧美国产精品一级二级三级| 亚洲国产精品国产精品| 肉色欧美久久久久久久蜜桃| 国产片内射在线| 亚洲av在线观看美女高潮| 麻豆av在线久日| 美国免费a级毛片| 日本欧美视频一区| 亚洲精品视频女| 色视频在线一区二区三区| 午夜精品国产一区二区电影| 香蕉丝袜av| av网站在线播放免费| 人妻系列 视频| 久久鲁丝午夜福利片| 十分钟在线观看高清视频www| 亚洲精品日本国产第一区| 午夜免费男女啪啪视频观看| 久久精品亚洲av国产电影网| 性色avwww在线观看| 久久精品熟女亚洲av麻豆精品| 只有这里有精品99| 热re99久久国产66热| 男人添女人高潮全过程视频| 久久99精品国语久久久| 久久 成人 亚洲| 久久国产精品男人的天堂亚洲| 亚洲精品久久久久久婷婷小说| 国产在视频线精品| 伦精品一区二区三区| 又粗又硬又长又爽又黄的视频| 亚洲精品中文字幕在线视频| 国产福利在线免费观看视频| 亚洲色图综合在线观看| 午夜福利视频在线观看免费| 日韩人妻精品一区2区三区| 青春草亚洲视频在线观看| 少妇精品久久久久久久| 超色免费av| 午夜91福利影院| 国产成人午夜福利电影在线观看| 一区二区av电影网| 99热全是精品| 国产av精品麻豆| 亚洲av电影在线观看一区二区三区| tube8黄色片| 69精品国产乱码久久久| www日本在线高清视频| 欧美黄色片欧美黄色片| 国产又色又爽无遮挡免| 国产1区2区3区精品| 80岁老熟妇乱子伦牲交| 爱豆传媒免费全集在线观看| 晚上一个人看的免费电影| 一级片免费观看大全| 久久韩国三级中文字幕| 亚洲视频免费观看视频| 中文字幕色久视频| 久久精品国产亚洲av天美| 亚洲av国产av综合av卡| 欧美日韩国产mv在线观看视频| 国产探花极品一区二区| 大片电影免费在线观看免费| 欧美成人午夜免费资源| 成人手机av| 老司机亚洲免费影院| 黄色配什么色好看| 97在线视频观看| 91成人精品电影| 欧美日韩一级在线毛片| 一级黄片播放器| 久久人妻熟女aⅴ| 国精品久久久久久国模美| 精品视频人人做人人爽| 十分钟在线观看高清视频www| 少妇熟女欧美另类| 色网站视频免费| 国产成人免费无遮挡视频| 亚洲精品美女久久av网站| 色哟哟·www| 高清在线视频一区二区三区| 国产黄频视频在线观看| 免费在线观看视频国产中文字幕亚洲 | 国产男女内射视频| 欧美亚洲日本最大视频资源| 久久99一区二区三区| 少妇猛男粗大的猛烈进出视频| 成人午夜精彩视频在线观看| 国产精品秋霞免费鲁丝片| 亚洲五月色婷婷综合| 中文字幕人妻丝袜制服| 精品第一国产精品| 久久婷婷青草| av免费在线看不卡| 伊人久久大香线蕉亚洲五| 欧美日韩成人在线一区二区| 超碰成人久久| 在线天堂最新版资源| 久久精品国产综合久久久| 免费在线观看完整版高清| 可以免费在线观看a视频的电影网站 | 2022亚洲国产成人精品| 精品久久久精品久久久| 一本一本久久a久久精品综合妖精 国产伦在线观看视频一区 | 精品人妻偷拍中文字幕| 高清不卡的av网站| 人妻一区二区av| 国产人伦9x9x在线观看 | 国产男人的电影天堂91| 老熟女久久久| av免费观看日本| 一二三四在线观看免费中文在| 国产av码专区亚洲av| 亚洲精品av麻豆狂野| 日韩在线高清观看一区二区三区| 国产一区亚洲一区在线观看| 国产欧美日韩一区二区三区在线| 成人毛片a级毛片在线播放| 丝袜美足系列| 老汉色av国产亚洲站长工具| 久久午夜综合久久蜜桃| 日韩欧美一区视频在线观看| 亚洲内射少妇av| 国产精品香港三级国产av潘金莲 | 一区二区日韩欧美中文字幕| 少妇人妻 视频| 一区二区三区激情视频| 亚洲激情五月婷婷啪啪| 色婷婷久久久亚洲欧美| 国产熟女欧美一区二区| 色播在线永久视频| 性高湖久久久久久久久免费观看| 久久久久久久久免费视频了| 黄片小视频在线播放| 欧美国产精品一级二级三级| 亚洲精品一区蜜桃| 久久精品国产自在天天线| 久久精品久久久久久久性| 视频在线观看一区二区三区| 老司机影院成人| 国产熟女欧美一区二区| 欧美日韩国产mv在线观看视频| 黄色一级大片看看| 亚洲精品av麻豆狂野| 免费高清在线观看日韩| 一本久久精品| 午夜福利在线免费观看网站| 日本av手机在线免费观看| 久久久欧美国产精品| 中文精品一卡2卡3卡4更新| 在线观看人妻少妇| 日韩制服骚丝袜av| 三上悠亚av全集在线观看| 日本av免费视频播放| 一边摸一边做爽爽视频免费| 一级,二级,三级黄色视频| 亚洲国产色片| 亚洲av日韩在线播放| 欧美国产精品va在线观看不卡| 人成视频在线观看免费观看| 男人爽女人下面视频在线观看| 欧美xxⅹ黑人| 男人爽女人下面视频在线观看| 男女下面插进去视频免费观看| 国产精品久久久久成人av| 看十八女毛片水多多多| 人人澡人人妻人| 久久精品aⅴ一区二区三区四区 | 啦啦啦在线观看免费高清www| 精品亚洲成a人片在线观看| 亚洲国产av新网站| 18禁观看日本| 99热国产这里只有精品6| 十八禁高潮呻吟视频| 美女国产视频在线观看| 亚洲国产精品国产精品| 精品一区二区三区四区五区乱码 | 女性生殖器流出的白浆| 久久ye,这里只有精品| 1024香蕉在线观看| 9色porny在线观看| 免费大片黄手机在线观看| 亚洲色图综合在线观看| 久久久久国产网址| 一区二区三区四区激情视频| 女性生殖器流出的白浆| 一级毛片我不卡| 亚洲av日韩在线播放| 成年人午夜在线观看视频| 永久免费av网站大全| a级毛片黄视频| 久久99一区二区三区| 欧美精品亚洲一区二区| 一级,二级,三级黄色视频| 欧美日韩精品网址| 天天躁夜夜躁狠狠久久av| 一二三四在线观看免费中文在| 日韩av在线免费看完整版不卡| 一二三四在线观看免费中文在| 午夜日本视频在线| 国产成人欧美| 99久久中文字幕三级久久日本| 久久狼人影院| 国产精品 欧美亚洲| 热99久久久久精品小说推荐| 国产亚洲最大av| 久久av网站| 精品少妇久久久久久888优播|