擁有好心情

藍正妹做的影片耶

好喜歡這首歌的旋律與歌詞



雨下在我窗前　玻璃也在流眼淚
街上的人都看起來比我幸福一點
用寂寞來測驗　還是最想要你陪
曾一起走過的夏天　我常常會夢見

﹡我猜不到你真正的感覺
　思念寫成臉上的黑眼圈
　有的時候我寧願　你對我壞一點
　無法停止幻想我們的永遠

＊愛你是孤單的心事　不懂你微笑的意思
　只能像一朵向日葵　在夜裡默默的堅持
　愛你是孤單的心事　多希望你對我誠實
　一直愛著你　用我自己的方式

我在你的心裡　有沒有一點特別
就怕你終究沒發現　我還在你身邊

From the JCBN/NC-IUB Newsletter 1981 [1]
Goldstein et al. [Nature (Lond.) 285, 86 (1980)] have presented the following definition and comments.

Definition

A lectin is a sugar-binding protein of non-immune origin that agglutinates cells or precipitates glycoconjugates.

Comments

1. A lectin molecule contains at least two sugar-binding sites; sugar-binding proteins with a single site will not agglutinate or precipitate structures that contain sugar residues, so are not classified as lectins.

2. The specificity of a lectin is usually defined by the monosaccharides or oligosaccharides that are best at inhibiting the agglutination or precipitation the lectin causes.

3. Lectins occur in many types of organism; they may be soluble or membrane-bound; they may be glycoproteins.

4. Sugar-specific enzymes, transport proteins and toxins may qualify as lectins if they have, multiple sugarbinding sites.

1. IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN), and Nomenclature Commission of IUB (NC-IUB),.Newsletter 1981, Arch. Biochem. Biophys., 1981, 206, 458-462; Eur. J. Biochem., 1981, 114, 1-4; Hoppe-Seyler's Z. Physiol. Chem., 1981, 362, I-IV; J. Biol. Chem., 1981, 256, 12-14.

gC(on the surface of viral envelope)--->


binds to the cell surface particle(heparan sulfate)

gD --->

binds specifically to the herpesvirus entry mediator receptor (HVEM)
providing a strong, fixed attachment to the host cell
(These interactions bring the membrane surfaces into mutual proximity and allow for other surface glycoproteins to interact)

gH.gL--->

Once bound to the HVEM, glycoprotein D changes its conformation and interacts with glycoproteins H and L, which form a complex
The interaction of these membrane proteins results in the hemifusion state

gB--->

glycoprotein B interaction with the glycoprotein H and L complex creates an entry pore.Glycoprotein B interacts with host cell surface glycosaminoglycans

Herpes simplex virus 1 and 2 (HSV-1 and HSV-2) are two strains of the herpesvirus family, Herpesviridae, which cause infections in humans.[1] HSV-1 and 2 are also referred to as Human Herpes Virus 1 and 2 (HHV-1 and HHV-2).

After an initial, or primary, infection, HSV establishes latency, during which the virus is present in the cell bodies of nerves which innervate the area of original outbreak. During reactivation, the virus is produced in the cell and transported outwardly via the nerve cell's axon to the skin.[2] The ability of HSV to become latent leads to the chronic nature of Herpes infection; after the initial infection subsides, Herpes symptoms may periodically recur in the form of outbreaks of herpetic sores near the site of original infection.

Herpes infections are marked by painful, watery blisters in the skin or mucous membranes (such as the mouth or lips) or on the genitals.[1] The blisters resemble those seen in chickenpox — an infection caused by a third member of the alpha-Herpesviridae subfamily, Varicella Zoster Virus (VZV), also known as Human Herpes Virus 3 (HHV-3). Lesions heal with a crudescent scab, the hallmark of herpetic disease. Herpes is contagious if the carrier is producing and releasing ("shedding") virus. This is particularly likely during an outbreak, although individuals may shed virus between outbreaks. Although no cure is yet available, treatments exist which reduce the likelihood of viral shedding. An HSV infection on the lips is commonly known as a "cold sore" or "fever blister" and should not to be confused with a canker sore; canker sores are not caused by the HSV virus.



Transmission
HSV is generally transmitted by direct contact of lips or genitals when the sores are present, or also when no sores are present (known as viral shedding). HSV can be present in semen, vaginal fluids, and saliva. In addition, herpes may be transmitted during childbirth, which can be fatal to the infant. The immature immune system of the child is unable to defend against the virus and even if treated, infection can result in brain damage. Transmission occurs while passing through the birth canal and the risk of infection is minimal if there are no symptoms or exposed blisters during delivery. The first outbreak after exposure to HSV is commonly more severe than future outbreaks, as the body has not had a chance to produce antibodies; this first outbreak also carries a low (~1%) risk of developing aseptic meningitis.



Cellular entry
Entry of HSV into the host cell involves interactions of several viral glycoproteins with cell surface receptors. The virus particle is covered by an envelope which, when bound to specific receptors on the cell surface, will fuse with the cell membrane and create an opening, or pore, through which the virus enters the host cell.

The sequential stages of HSV entry are analagous to those of other viruses. At first, complementary receptors on the virus and cell surface bring the two membranes into proximity. In an intermediate state, the two membranes begin to merge, forming a hemifusion state. Finally, a stable entry pore is formed through which the viral envelope contents are introduced to the host cell.[3]

In the case of Herpes virus, initial interactions occur when glycoprotein C, on the surface of the viral envelope, binds to a cell surface particle, heparan sulfate. Glycoprotein D binds specifically to the herpesvirus entry mediator receptor (HVEM), thus providing a strong, fixed attachment to the host cell. These interactions bring the membrane surfaces into mutual proximity and allow for other surface glycoproteins to interact.

Once bound to the HVEM, glycoprotein D changes its conformation and interacts with glycoproteins H and L, which form a complex. The interaction of these membrane proteins results in the hemifusion state. Afterward, glycoprotein B interaction with the glycoprotein H and L complex creates an entry pore.[3] Glycoprotein B interacts with host cell surface glycosaminoglycans.




Genetic inoculation
After the viral capsid enters the cellular cytoplasm, it is transported to the cell nucleus. Once attached to the nucleus at a nuclear entry pore, the capsid ejects its DNA contents via the capsid portal. The Herpes capsid portal is a ring containing twelve (12) of the same protein, Herpes Capsid Portal Protein, produced by the herpes gene UL-6.[4] The DNA exits the capsid in a single linear segment. [5] The viral DNA enters the nucleus via the pore. Once the DNA has entered the nucles, replication may begin.



Replication
Consequent to a cell being infected, groups of Herpes virus proteins, termed immediate-early, early, and late proteins, are produced following specific time periods. Research using a new flow cytometry methodology in KSHV indicates the possibility of an additional lytic stage, delayed-late.[6] These stages of lytic infection, particularly late lytic, are distinct from the latency stage. For example, in the case of HSV-1, no protein products are detected during latency.

Upon entering the cell, an α-TIF protein also joins the viral particle and aids in immediate-early Transcription. The virion host shutoff protein (VHF-UL41) is very important to viral replication. This enzyme shuts off protein synthesis in the host, degrades host mRNA, helps in viral replication, and regulates gene expression of viral proteins. While the viral genome immediately travels to the nucleus, the VHF protein remains in the cytoplasm. The packaging of the viral particles, which include the genome, core and the capsid, occur in the nucleus. In the nucleus, cleavage of genome concatemers occurs and these are placed into pre-formed capsids. The viral envelope is acquired from the nuclear envelope, more specifically the inner lamellae of the membrane.[7]



Latent infection
HSV may persist in a quiescent but persistent form known as latent infection, notably in neural ganglia.[1] During latent infection of a cell, HSV express Latency Associated Transcript (LAT) RNA. LAT is known to regulate the host cell genome and interferes with natural cell death mechanisms. By maintaining the host cells, LAT expression preserves a reservoir for the virus, which allows later recurrences to produce further infections.

A protein found in neurons may bind to Herpes DNA and regulate latency. Recent studies have found that the Herpes DNA contains a sequence that is involed in silencing the expression of a gene associated with lytic infection, ICP4. The sequence contains elements which bind to human nerve cell protein factors: the human neuronal protein Neuronal Restrictive Silencing Factor (NRSF), and human Repressor Element Silencing Transciption Factor (REST). When the proteins are able to bind to the viral DNA elements, histone deacytalization occurs atop the ICP4 gene sequence.

一般分子物質能於polyacrylamide gel中經電泳而被分離，並可再將其由膠體轉印到硝化纖維膜(nitrocellulose membrane)上相對的位置，然後再利用抗血清與蛋白質作專一性結合進行免疫染色；此過程稱之西方氏轉漬法(Western blotting) 或電泳轉漬法(electroblotting)。此方法可用於對未知的抗原或抗體作性質上的分析、確定血液或組織中是否有細菌性抗原存在、及檢測曾經曝露在病原菌的個體之血清是否呈陽性反應。一般西方氏轉漬法分析的抗原以蛋白質抗原為最典型；至於其他大分子亦可被分離與轉印至硝化纖維膜進行分析，如：醣蛋白(glycoprotein)、脂質蛋白(lipoprotein)、醣脂質(glycolipids)及脂質聚醣物(lipopolysaccharides)。

　　西方氏轉漬法最主要的優點是對不同的抗原具有高度的專一性。臨床上，此技術經常與其他的免疫分析法(如ELISA)配合使用，以確定是否有錯誤陽性反應的情形。然而此方法亦有缺點，首先西方氏轉印法主要是定性的方法，若要進行抗體或抗原的定量分析則有困難；其次，抗原經由SDS-PAGE電泳分離後會被變性(denatured)，可能會減少或破壞其抗原活性(antigenic activity)，造成抗體無法辨識。因此，研究者要以此技術進行實驗時，必須考慮各種最適合的條件。首先要決定膠體百分比以利待測的抗原具有最佳解析力；接著必須決定抗原是否能夠結合在硝化纖維膜上；最後確定整個抗原測定的過程為高專一性及高靈敏度的。




Western blotting（西方墨點轉漬法）的免疫檢驗方法，是可以強力偵測一細胞或體液中特定的微量蛋白質，這項技術是結合膠體電泳（gel electrophoresis）的優越解析力、抗體的專一性、enzyme的敏感性。如此一來，使得在一複合物中，海底撈針地找尋一蛋白質，成為可能。

Western blotting又稱immunoblotting，主要分成三個步驟：

第一步，將蛋白質混合物置於SDS-polyacrylamide膠體上，電泳分離，接著是blotting：解析的蛋白質在電場中，從膠體轉移到Nitrocellulose transfer membrane。

第二步，將transfer membrane浸於含有標的蛋白質的抗體（primary antibody）溶液中，形成抗原-抗體複合體。

第三步，藉由對primary antibody具專一性的enzyme-linked抗體（secondary antibody），來漂洗此membrane，再加入受質，產生有色物質，而偵測得知感興趣蛋白質帶。

（1）細胞的準備與處理

1. 本實驗是以Wistar rats做為實驗動物，藉由脊髓管注射藥物於脊髓腔內，建立嗎啡耐藥性試驗動物 模式。再依照既訂的時間將Wistar rats斷頭並快速取出新鮮脊髓，除去硬腦膜（dura mater）並避免血液污染脊髓，最後截取spinal segments L3-5，並分成同側與對側（ipsilateral and contralateral sides）之背角與腹角（dorsal and ventral horns），放置於-80℃冰箱保存，以待後續脊髓蛋白質的分析。

（2）蛋白質的萃取

1. 將lysis buffer 300μl（內含protinase inhibitor）加入放置於4℃冰鎮之脊髓標本Ependrof tube，並以Microson機器打碎及sonication標本成均質液體，再用超高速離心方法（4℃下以65000至70000 rpm之速度，離心35分鐘）；其後分離出上清液與沉澱物，丟棄沉澱物，留下上清液。

2. 此上清液（含蛋白質），以modified Bradford method定量蛋白質含量（以Bio-Tek Elx800分析吸光值，來測定各標本之蛋白質量）。

3. 將經校訂後取等體積與等蛋白質總量之標本（50或100μl），置入sample collecting tubes內，再加入同體積的Tricine SDS sample buffer，於95℃下加熱15分鐘後，於4℃下離心（7000rpm），等待應用loading在SDS-PAGE gel上。

（3）Tricine Sodium Dodecyl Sulfate（SDS）膠體之準備

1. 配置7﹪的分離膠體（separation gel for NOS）：將30﹪Acrylamide solution 3.5ml，DDW 1.5ml，3M pH8.45 Tris –HCl 5ml，40﹪glycerol 5ml，10﹪Ammonium persulfate 75μl及TEMED 7.5μl混合均勻後；立即注入注膠器之玻璃板間，並於其上層注入100﹪酒精，使其表面平整，靜置至膠體硬化後，再將上層酒精倒出吸乾。

2. 配置10﹪的分離膠體（separation gel for COX-2）：將30﹪Acrylamide solution 5ml，3M pH8.45 Tris –HCl 5ml，40﹪glycerol 5ml，10﹪Ammonium persulfate 75μl及TEMED 7.5μl混合均勻後；立即注入注膠器之玻璃板間，並於其上層注入100﹪酒精，使其表面平整，靜置至膠體硬化後，再將上層酒精倒出吸乾。

3. 再注入配置好的5﹪聚積膠體（Stacking Gel：30﹪Acrylamide solution 0.5ml，3M pH 8.45 Tris –HCl 1.25ml，DDW 2ml，10﹪Ammonium persulfate 25μl及TEMED 5μl），於分離膠體上層，迅速插入0.75mm，10 wells之comb，來製備樣品槽，之後靜置至膠體凝固。

4. 完全聚合後，小心將comb拔出，再將sandwich clamp assemblies放入電泳槽中，加入10X Tricine SDS/electrophoresis buffer（Tris base 121g，Tricine 179g，SDS 10g，add DDW to final 1000ml），觀察無洩漏情況後，即完成膠體與電泳槽之製備。

（4）加入樣本（Loading sample）及電泳

1. 樣品槽由左至右，依序注入已染色之分子量標定蛋白質（pre-stained molecular weight marker），及其餘製備之脊髓樣本液，以進行電泳。

2. Tricine SDS/electrophoresis buffer solution要加入電泳槽內，先倒入正極槽，再倒入負極槽，注意不要有氣泡黏在膠體上。

3. 電壓在前十分鐘（聚積層）用80 volts，帶樣本移動到分離層再調高到125 volts。

4. 待tracking dye跑超出底層並且marker可看到呈現（210、105與70Kd清楚的分開），此時關掉電源，結束電泳，隨即拆除電泳儀組，小心取出膠體，浸入TTBS（Tris-Tween buffer saline）約一分鐘之後，進行轉印（electrotransfer）步驟。

（5）轉印（electrotransfer）步驟

1. 先將nitrocellulose（NC）paper（transfer membrane）及3M濾紙浸泡於轉印緩衝液（pH10.4，Transfer buffer：CAPS 2.2g、methanol 200ml、DDW 800ml）中，直到紙之各部分均勻潤濕為止。

2. Electrotransfer的方向是由負極（anode）到正極（cathode），而NC paper要置於接近正極，膠體那一面向負極。

3. 將已浸濕的3M濾紙，整齊重疊至於半乾式電轉漬器中間，為最底層。再平舖膠體於濾紙之上，並將NC paper蓋在膠體上，最上層再舖上3M濾紙，最後蓋上蓋子。接上電源，維持電流130mA，轉印overnight（至少14至15小時）。

（6）免疫轉漬法（immunoblotting）

1. 轉印完成後，將NC paper置於3﹪Blot-Quick blocking solution中，用迴旋振盪器振盪三十分鐘blocking。

2. 加入初級抗體（COX-2與nNOS antibody），用迴旋振盪器振盪作用二至三小時。

3. 以TTBS（Tris-Tween buffer saline）強力振盪洗三次，每次八分鐘。再將transfer membrane置於3﹪Blot-Quick blocking solution中，用迴旋振盪器振盪三十分鐘blocking。再加入次級抗體（1：500 polyclonal anti-rabbit IgG for COX-2，monoclonal anti-mouse IgG for nNOS），作用50分鐘。

4. 之後以TTBS強力振盪洗三次，每次八分鐘。

5. 再將transfer membrane夾起蔭乾，置入chemiluminescence reagents（PerkinElmer Life Science；Western Lightning：Enhanced luminol reagent 1.5ml配上Oxidizing reagent 1.5ml）作用3分鐘反應螢光。

6. 最後以Kodak之X-ray壓片，洗片後觀察結果。

gC-1-null virus：病毒不表現gC

ΔgC2-3: gC有缺陷的病毒

VP16:VP16 is a herpes simplex virus (HSV)-encoded transcriptional activator protein that is essential for efficient viral replication and as such may be a target for novel therapeutic agents directed against viral gene expression.

http://mcb.asm.org/cgi/content/abstract/14/5/3484

lectin:http://zh.wikipedia.org/wiki/%E5%87%9D%E9%9B%86%E7%B4%A0