【整形風 Medi Life 蔡豐州醫師】
   
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PHD
文章出處
  蔡豐州醫師
日期&閱覽
 張貼日期:2005/8/4  閱覽次數:10082

--- What inferences can we draw from these facts?

PHD
@
1, Fibroblasts were defined by positive immunohistochemical staining for D7-FIB (fibroblast marker) (Serotec, Oxford, UK), vimentin (mesenchymal cell marker), and collagens I and III, and negative staining for endothelial (von Willebrand factor (vWF)), mesangial (desmin) and epithelial (cytokeratin) cell markers
2, undifferentiated "reserve cell fibroblast" ---stain for
the human progenitor cell antigen CD34. These cells are known as dendritic interstitial cells and they richly populate collagenous connective tissues-normal  areolar, dermal, and visceral connective tissues, particularly inthe adventitia of vessels and skin adnexae. 
3, activated more functional fibroblast, use procollagen (collagen type I) antibody. 
4, myofibroblasts--- use the 1A4 clone of alpha smooth muscle
actin.
It is a theory that the CD34+ cells differentiate into the latter
two types during tissue remodeling and in neoplasia.
5, As CD34 is described as a stem cell marker and it being unlikely to be expressed on differentiated cells,
6, anatomic-site specific phenotype: 如 Chen B, et al IL-1 beta induces IL-6 expression in human orbital fibroblasts: identification of an anatomic-site specific phenotypic attribute relevant to thyroid-associated ophthalmopathy. J Immunol. 2005 Jul 15;175(2):1310-9.
@ 蛋白質或基因傳遞工具:
(1) Retrovirus-based delivery system
    --- 1, oncornaviruses 2, lentiviruses eg, HIV 約可以乘載8kb的cDNA片段 3, spumaviruses
(2) Adenovirus-based delivery system
    --- 1, linear, double-stranded DNA virus
         2, 會引起免疫反應(high-immunological)
(3) Electroporation
    --- 1, Wong & Neumann等人於1982年利用電場造成細胞膜的通透性改變
         2,  較適合於懸浮細胞株
(4) Microinjection
(5) Lipid-based delivery system
    --- 1, 利用帶正電的微脂粒包裹帶負電的DNA形成micelle, 再藉由endocytosis送入細胞內
         2, 容易為endosome所分解
         3, 目前成功率只有40 to 70 %
(6) Calcium-Phosphate transduction
    --- 1, 1973年由Graham & van der Eb等人發現
(7) Protein transduction technology
    --- 1, <30 a.a.的PTDs(protein transduction domains)可以攜帶異源的蛋白質結構, 以開展的型態直接加入細胞培養液中, 並於短時間(<15mins)進入細胞內
         2, in vitro, in vivo都有很高的傳遞率(-100%)
         3, antigenetic很弱
         4, 應用於攜帶異源蛋白質的PTDs有: 
             1, HIV-1的基因產物(trans-activator, Tat, YGRKKRRQRRR)
                    
             2, Drosophila果蠅的Antennapedia(ANTp, RQIKIWFQNRRMKWKK)
              上述它們都富含Arginine, Lysine, 在生理狀態的PH值, 帶高度正電的蛋白質胜肽鏈. 利用此帶正電的特性, 可以和細胞表面帶高度負電, 屬於GAG(glycosaminoglycan)的Heparan sulfate之間相互作用後, 以開展的蛋白質型態(denatured form)快速通過細胞膜, 而進入細胞. 進入之後由chaperone幫助摺疊, 完成正確的蛋白質結構, 執行其正常的生物功能

@ PGE2
    GLUCOSTEROID: Glucocorticoid Inhibition of Fibroblast Proliferation and Regulation of the Cyclin Kinase Inhibitor p21Cip1  (Molecular Endocrinology 11: 577–586, 1997)
Glucocorticoids, on the other hand, inhibit fibroblast proliferation and delay wound healing. The mechanism of glucocorticoid inhibition of fibroblast proliferation is unclear, and the data on the effects of glucocorticoids on fibroblast proliferation in culture are sometimes conflicting. In general, low concentrations of glucocorticoids potentiate the mitotic stimulation that occurs when growth factors are added to serum-starved fibroblasts. On the other hand, higher concentrations of glucocorticoids inhibit proliferation of mid-log phase cultures of fibroblasts.

Naturally occurring and synthetic glucocorticoids inhibit proliferation of mouse L929 fibroblasts in culture. Addition of 10-7 M dexamethasone to mid-log L929 culture causes an accumulation of cells with a G1 DNA content. Similar results have been observed in glucocorticoid-treated lymphoid cells. Inhibition of G1 progression in lymphoid cells is due to the effects of glucocorticoids upon G1 cyclin gene expression, and we have undertaken to test the hypothesis that a similar mechanism prevails in fibroblasts.
Progression through the G1 phase of the cell cycle is regulated by a family of serine/threonine protein kinases called cyclin-dependent kinases or Cdks, which are activated by association with a stimulatory subunit, a cyclin. Two families or classes of cyclins interact with at least 3 different Cdks during G1 progression: members of the D cyclin family (cyclin D1, cyclin D2, and cyclin D3) bind to and activate Cdk4 and Cdk6, whereas cyclin E activates Cdk2.

Inhibition of Cdk activity is mediated, in part, by a second set of regulatory subunits, the cyclin kinase inhibitors (reviewed in Refs. 14–16) There are 2 distinct classes of cyclin kinase inhibitors. Relatives of INK4 bind to Cdk4- and Cdk6-containing complexes, causing dissociation of the cyclin and inhibition of kinase activity. One of these inhibitors, p15INK4B, is thought to play a role in TGFß inhibition of keratinocyte proliferation. A second family of cyclin kinase inhibitors is comprised of relatives of p21Cip1. Members of this family bind to Cdk2-, Cdk4-, and Cdk6-containing complexes, prevent their activation at low stoichiometry, and block kinase activity at high stoichiometry. Induction of p21Cip1 is involved in p53-mediated cell cycle arrest, in proliferative senescence, in replication and repair of damaged DNA, and during differentiation of specific tissue types. A relative of p21Cip1 called p27Kip1 is thought to be involved in contact inhibition, cAMP inhibition of cell proliferation, and TGFß inhibition of epithelial cell proliferation.

It is believed that cyclin kinase inhibitors block cell proliferation by virtue of their ability to inhibit Cdk-dependent phosphorylation of critical substrates in the G1 phase of the cell cycle. Both Cdk2 and Cdk4 can phosphorylate tumor suppressor gene products, including the product of the Rb-1 gene, pRb, and the related protein p107. In cells that express wild type pRb, phosphorylation of pRb is necessary for initiation of S phase, and cells that harbor Rb-1 mutations do not require cyclin D for G1 progression. The preponderance of evidence indicates that phosphorylation of tumor suppressor gene products by cyclin D/Cdk4 may be central to the role of these kinases in regulating cell cycle progression. Cyclin E/Cdk2 kinase probably plays a role in activating E2F-like transcription factors which, in turn, control nucleotide metabolism in late G1 and S phase.

The D type cyclins, in conjunction with Cdk4 or Cdk6, appear to be primary targets for hormones that stimulate cell proliferation. D type cyclins are induced by a large number of growth factors, and abrogation of D cyclin induction blocks the mitogenic effects of such hormones. Hormones that inhibit cell proliferation, such as TGFß, cause a decrease in the amount or activity of cyclin D/Cdk4 complexes. Glucocorticoid inhibition of lymphoid cell proliferation is due, in part, to inhibition of cyclin D3 expression. We speculated that a similar mechanism might account for glucocorticoid inhibition of fibroblast proliferation.

pentosan polysulfate(PPS)
間質性膀胱炎(interstitial cystitis, IC)症狀大多是以頻尿與骨盆腔疼痛等症狀表現為主,通常這些症狀與泌尿道感染、尿道炎等疾病所引起的症狀相似而不易判斷。治療上大多採用症狀療法或肝素(heparin)膀胱灌注等方式,以緩解病患疼痛,改善排尿功能。美國FDA在1996年核准pentosan polysulfate(PPS)(商品名 Elmiron®)上市,用來緩解間質性膀胱炎引起的疼痛與不適感,使得PPS成為第一個,也是現今唯一以口服方式治療IC的藥物。
作用機轉
PPS的結構類似heparin(圖一),因此PPS具有抗凝集(anticoagulant)與纖維蛋白溶解(fibrinolytic)的作用2。抗凝集作用可能與antithrombin III有關,但是只有heparin的抗凝集作用的十分之一。另外因刺激內皮細胞釋放出tissue-type plasminogen activator而產生纖維蛋白溶解作用,基於上述作用,目前臨床上正在研究PPS用於深部靜脈栓塞的預防效果。而在治療IC時似乎是藉由另外的機轉,因為引起IC的原因尚未完全明瞭,有一種解釋認為IC是因為膀胱壁內膜的成分-氨基葡萄聚醣(glucos-amino-glycans, GAG)的缺乏或受損,造成尿液中的細菌、有毒物質以及鉀離子穿透GAG層,刺激膀胱壁,因而導致疼痛,影響排尿功能3,4。PPS的功能類似GAG,可以強化(augment)受損的內膜GAG層,減少有毒物質對GAG的穿透。另外PPS也有抑制膀胱發炎反應的作用,對IC的症狀也有些許助益。
藥物動力學
PPS口服吸收率約3﹪,主要代謝器官為肝臟與脾臟,半衰期是4.8小時,由尿液排除。
適應症與用法
FDA目前核准的適應症只有IC,用法為每天三次,每次100 mg。通常服用2至4週後治療效果開始出現,一般以三個月為一個療程。若服用完三個月後症狀沒有改善,可再服用三個月。連續服用6個月後無法得到療效,則不建議繼續服用。臨床研究顯示約有30﹪的病人服用PPS後症狀可得改善8,9。與對照組相比,對於疼痛緩解、排尿急迫感與頻尿的次數可分別降低37﹪、28﹪與54﹪10。
不良反應
發生率1~10﹪的不良反應有頭痛、頭暈(dizziness)、禿頭(alopecia)、皮膚疹、腹瀉、噁心、消化不良(dyspepsia)、腹痛與肝功能指數異常。小於1﹪發生率較嚴重不良反應如貧血、延長凝血西每原時間(prothrombin time)、白血球低下(leukopenia)與血小板低下(thrombocytopenia)7。
交互作用
PPS目前沒有和其他藥物有明顯的交互作用,但是在與其他抗凝血藥物或是水楊酸類藥品同時使用時,要注意可能有增加出血的機率,在使用這些藥物前應先告知醫師目前正在服用PPS。服藥後若有出血現象(例如血尿或黑便、突然出現瘀傷、牙齦出血等)請立刻與醫師聯絡。
結 論
PPS屬於低分子量肝素的一種,一樣具有抗凝集的作用。對於間質性膀胱炎的療效則是藉由強化膀胱內壁的GAG層,減少有毒物質對膀胱的影響,進而緩解疼痛。與肝素膀胱灌注(每週三次)相比,病患接受度較高,也相當方便。對於其他方面的應用,如預防深部靜脈栓塞,則需大型臨床試驗來證實療效。

@



@ Development:

Yun-Jin JIANG 江運金
Principal Investigator
Laboratory of Developmental Signalling and Patterning
Institute of Molecular and Cell Biology,
Proteos, 61 Biopolis Drive,
Singapore 138673
Tel: (65) 65869718 (office)
(65) 65869715 (lab)
Fax: (65) 67791117
e-mail: yjjiang@imcb.a-star.edu.sg
The mesoderm gives rise to tissues including connective tissue, muscles, and the circulatory system. The mesoderm is also believed to be responsible for the formation of the central nervous system. For example, the notochord is responsible for releasing certain factors which induce the ectoderm to become neural tissue.

In a developing vertebrate embryo, the mesoderm differentiates into these areas:
1, The chordamesoderm lies along the central axis, under the neural tube, and gives rise to the notochordal process which later becomes the notochord.
2, The paraxial mesoderm, at the sides of the neural tube, gives rise to the somites and head mesoderm. The somites form the vertebral column dermis and skeletal muscle, while the head mesoderm will develop into facial muscle and cartilage.
3, The intermediate mesoderm, between the paraxial mesoderm and the lateral plate, develops into the part of the urogenital system (kidneys and gonads).
4, The lateral plate mesoderm is found at the periphery of the embryo. It will, in turn, split into two layers, the somatic layer/mesoderm and the splanchnic layer/mesoderm. Enclosed between the two is a space called coelom. The somatic layer forms the future body wall, and the splanchnic layer forms the circulatory system and future gut wall.

(1) Pattern formation is the fundamental process, which ensures that an organism develops into the highly stereotypic array of different cells and tissues that is characteristic for a species. Pattern formation occurs in a two-dimensional plane, with help of the two existing axes, the antero-posterior (A/P) and dorso-ventral (D/V) axes
Mesodermal patterning. The embryonic mesoderm is specified during gastrulation, with dorsal mesoderm becoming notochord, lateral (paraxial) mesoderm forming muscle, and ventral mesoderm becoming blood. Although fate mapping experiments show that mesodermal cells become restricted to specific fates during early gastrulation, transplantation experiments show that fates are not fixed until later. Thus, local cell-cell interactions are critical for reinforcing cell fate decisions. spadetail (spt), a gene encoding a T-box transcription factor, is required for proper gastrulation movements and fate specification of paraxial mesodermal (somitic) cells, whereas no tail (ntl), a gene encoding another T-box family member, is critical for development of the axial (notochordal) mesoderm. Together, the two T-box genes are important for the development of all mesodermal types.
Mesodermal segmentation. Following gastrulation, the paraxial mesoderm becomes visibly segmented into a reiterated series of tissue blocks called somites. Somitogenesis involves cell-cell interactions that convert a field of identical cells into a set of individual compartments, each of which is patterned along the anterior to posterior axis. spt acts near the top of the pathway to get cells to the right place at the right time.

a genetic screen to identify mutations that disrupt segmental gene expression of her1, a homolog of the Drosophila pair-rule gene hairy.

(2) Two ligands of the Notch receptor exist in Drosophila, encoded by the Delta (Dl) and Serrate (Ser) genes. The activity of the Notch receptor is dependent not only on the presence of one of these ligands, but also on the ratio of the concentration of ligand versus receptor. If the ratio is below a certain value, Notch is activated, if above, the activation of the receptor is suppressed in a cell autonomous manner.
The Notch receptors are large transmembrane proteins that are bound and activated by a family of related ligands, the Delta–Serrate–Lag2 (DSL) proteins. Activation of the receptors leads to a proteolytic liberation of the intracellular domain (ICD), which translocates into the nucleus and forms a complex with an evolutionarily conserved, DNA-binding protein,referred to as Su(H) but also known as CBF-1, RBP-Jk, and lag-1 in various species.
Notch ICD converts Su(H) from a transcriptional repressor to a transcriptional activator of Notch target genes.
somite defects are seen in mouse embryos with mutations in the
Notch1 receptor, the Notch ligands, Dll1 or Dll3, or in the mouse homolog of Su(H) (Conlon et al. 1995; Oka 1995; de Angelis et al. 1997; Kusumi et al. 1998). Similar somite defects are generated in Xenopus embryos when the Notch signaling pathway is blocked by ectopic expression of dominant-negative forms of X-Delta-2, or of
Xenopus Su(H) [X-Su(H)] (Jen et al. 1997)
the molecular clock in the form of c-hairy1 expression(chicken) to the periodic activation of the Notch pathway within the presomitic mesoderm via lunatic fringe.
two novel WRPW–bHLH genes, called ESR-4 and ESR-5, whose expression in the paraxial mesoderm is activated by the Su(H)-dependent Notch pathway. The expression of ESR-4/ESR-5 indicates that the Notch signaling pathway is activated in a broad domain of cells in the tailbud, but resolves into an ON/OFF state corresponding to A/P half-segments, respectively, when paraxial cells initiate segmentation.
(3) Shh (Sonic Hedgehog):
3a, http://www.learner.org/channel/courses/biology/units/gendev/images.html
3b,
Introduction
Cell-cell signaling is a crucial aspect of development and yet just five signal transduction pathways mediate the early development of most animals. These intercellular signaling pathways consist of: Wnt, TGF-ß, Notch, RTK (receptor tyrosine kinase) and Hedgehog pathways.1 The most common target of signaling in development is transcription. Different pathways activate or repress different genes at distinct times and places in the embryo.1 Signaling pathways have important roles in determining embryonic patterning and cell fate decisions. Analysis of Drosophila development has been vital in elucidating the components and functions of these signaling pathways. Research in vertebrates revealed that not only are the same signaling components also found, but often the developmental roles were similar to those in Drosophila. This review will focus on one of the vertebrate Hedgehogs, Sonic hedgehog, by addressing its signaling mechanism and roles in vertebrate development.

Hedgehog Ligands
The vertebrate hedgehog family is represented by at least three members: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). The original hedgehog gene, identified in Drosophila, is named after its mutant phenotype: the embryo is covered with pointy denticles, resembling a hedgehog. Two vertebrate genes are named after species of hedgehogs (dhh, ihh) and shh after a video game character. Shh is the most extensively characterized vertebrate homolog, and is involved in a wide variety of embryonic events. It can act as both a short-range, contact-dependent factor and as a long-range, diffusible morphogen.2,3 Shh genes are highly conserved and have been identified within a variety of species, including human, mouse, frog, fish, and chicken. Mouse and human Shh proteins are 92% identical at the amino acid level.4 In the human embryo, Shh is expressed in the notochord, the floorplate of the neural tube, the gut, and in the developing limbs.5


Fig. 1. Shh undergoes autocatalytic processing prior to secretion. The Shh precursor protein is cleaved to yield an ~20 kDa N-terminal domain (signaling domain) and an ~25 kDa C-terminal domain (catalytic domain). Cholesterol modification is important for secretion and activity of the Shh protein. [Note: figure is adapted from reference 7.]

Hedgehog proteins undergo autocatalytic processing and modification that is critical for signaling activity (see Figure 1). The ~45 kDa precursor protein is cleaved to yield an ~20 kDa N-terminal domain and an ~25 kDa C-terminal domain.6 Autoprocessing of Hedgehog also causes the covalent attachment of cholesterol onto the carboxy terminus of the N-terminal domain. The N-terminal domain retains all known signaling capabilities while the C-terminal domain is responsible for the intramolecular precursor processing, acting as a cholesterol transferase.6 The cholesterol moiety is thought to direct Hedgehog protein traffic in the secretory cell.7 Another form of human Shh is also produced by mammalian and insect cells. This form contains both cholesterol and palmitoyl groups. The site of the palmitic acid modification was identified at Cys-24, on the N-terminal domain.8 These lipid modifications do not affect binding affinity of Shh to the Patched-1 receptor but do significantly enhance the activity of Shh protein in the C3H10T1/2 alkaline phosphatase assay.8 Thus, cholesterol may be important in the Hedgehog pathway not only for signal generation, affecting secretion, but also for signal reception, affecting activity.9

Signaling Pathway
The canonical Hedgehog signaling pathway is a tale of two transmembrane proteins (see Figure 2). Patched (Ptc), a twelve-pass membrane protein binds Hedgehog ligand. Smoothened (Smo), a seven-pass membrane protein is a signal transducer. In the absence of ligand, Ptc interacts with and inhibits Smo, either directly or indirectly. This repression culminates in a transcription factor acting as a transcriptional repressor. The transcription factor is called Cubitus interruptus (Ci) in Drosophila and Gli in vertebrates. There are 3 Gli genes in vertebrates, each with distinct transcriptional functions. When Hedgehog binds Ptc, Ptc's interactions with Smo are altered such that Smo is no longer inhibited. This leads to Ci/Gli protein entering the nucleus and acting as a transcriptional activator for the same genes it represses when Ptc is free to interact with and inhibit Smo. Studies in vertebrates indicate that the determination of diverse cell fates by Shh signaling occurs by regulating the combination of Gli genes expressed in a cell (for reviews, see references 7 and 10-12).


Fig. 2. The Shh signaling pathway involves two transmembrane proteins, Patched (Ptc) and Smoothened (Smo). Ptc binds Shh, whereas Smo acts as a signal transducer. In the absence of ligand, Ptc interacts with and inhibits Smo. This inhibition activates a transcriptional repressor (e.g. Gli in vertebrates). In the presence of ligand, the interaction of Ptc and Smo is altered and Smo is no longer inhibited. Gli protein may then enter the nucleus and function as a transcriptional activator. [Note: figure is adapted from reference 7.]

This model of Hedgehog signal transduction was predominantly worked out in Drosophila, where mutational analysis is easier and faster than in mammals; however, the model is holding up well in vertebrate studies. Binding of Ptc to Smo has been demonstrated for vertebrate homologs, although it is unknown whether binding is direct, since it was observed in the context of other cellular proteins.12 Mammals may express at least two Patched proteins, Patched-1 (Ptc-1) and Patched-2 (Ptc-2), both of which bind vertebrate Hedgehogs with similar affinity.13 As expected from the model, mice lacking Ptc-1 are lacking the inhibition of Smo activity and have constitutively activated Shh response genes in target tissues.14 Ptc-2 is likely to play a role in Dhh signaling as the expression patterns are similar, but a mutant Ptc-2 phenotype has not been reported.13 Analysis of Smo null mutant mice reveals phenotypes identical to Shh mutants and additional phenotypes not associated with Shh. These results indicate that Smo activity during development is likely not limited to activation of the Shh pathway.15 Interestingly, in Drosophila, Hedgehog-independent Ptc function and Ptc- independent Hedgehog function have also been observed.10 Thus, the Hedgehog signaling pathway may be more complicated than the canonical model suggests, as it is likely that pathway components interact with additional ligands and receptors that have yet to be identified.

Long- and Short-range Signaling
In flies and vertebrates, Hedgehog ligands can act either in short-range or long-range signaling. Both short and long-range signaling mechanisms are mediated by the N-terminal peptide after autoprocessing.16 Due to the addition of lipid moieties during processing, Shh N-terminal peptide is localized to the cell membrane.17 Short-range signaling events, over 1 or 2 cell diameters and in a contact-dependent manner, are easier to reconcile with a membrane-associated ligand. How then does the lipid-modified Shh mediate long-range signaling, where different concentrations of Shh induce different cell fates? Three mechanisms have been proposed to explain the long-range effects of Shh: simple diffusion of Shh; a relay mechanism in which Shh activates secondary signals; and direct delivery of Shh through cytoplasmic extensions.18

Cell culture and in vivo studies support the simple diffusion model of long-range signaling. Initial studies using a soluble, N-terminal processed form of Shh demonstrate that it is sufficient to direct long-range effects in vertebrate motor neuron induction in chick embryos.19 Although this soluble form lacks lipid modifications, a recent report provides evidence that a native, cholesterol-modified form of Shh is biologically potent and mediates long range signaling.18 Soluble, lipid processed Shh (s-ShhNp) is formed by concentrating cholesterol-modified, processed Shh into lipid rafts, where it multimerizes with the lipid attachments on the inside of the multimer. This soluble form of Shh can be isolated from chick limb buds, a biologically relevant source of Shh. In such tissues, s-ShhNp is thought to be freely diffusible and able to form a gradient to facilitate long-range signaling.18 Likewise in the mouse limb bud, cholesterol modification is essential for the normal range of signaling.20 Further studies in the chick neural tube using a deleted form of Ptc-1 to block Hedgehog signals, also support a gradient mechanism where Shh acts directly and at long-range.21 Importantly, visualizing Shh at a distance from sites of expression has now been achieved. Using optimized immunohistochemistry techniques in the mouse, Shh ligand is detectable over considerable distances depending on the tissue. Visualization of Shh proteins in target tissues is achieved under conditions allowing proteoglycan/glycosaminoglycan (PG/GAG) preservation, suggesting a role for PG/GAG in controlling Shh movement during long-range signaling.22 These results all support the model of direct, diffusible, long-range action by Shh.

Shh in Vertebrate Development
The Shh signaling pathway functions throughout development. Shh is involved in the determination of cell fate and embryonic patterning during early vertebrate development. An example of this activity is the patterning of the neural tube such that motor neurons are derived from the ventral region and sensory neurons are formed from the dorsal region.10 Later in development, Shh is involved in the proper formation and function of a variety of tissues and organs. Table 1 shows a list of Shh-dependent organs and tissues in vertebrate development. It should be noted that in some cases Shh works with other signaling factors such as FGFs, Wnts, and BMPs to mediate developmental processes.

Vertebrate embryonic development utilizes both short- and long-range mechanisms of Shh signaling. Short-range signaling by Shh is apparent during floor plate induction by the notochord within the neural tube.2 Long-range signaling by Shh occurs during motor neuron formation in the neural tube, sclerotome induction and proliferation in the somites, and limb patterning along the anterior-posterior axis.23 These developmental events were some of the first to be characterized for Shh signaling. In other embryonic patterning events where Shh is known to have activity, it is still unclear whether signaling acts locally or at a distance from Shh-producing cells. For this review, only several of the multiple developmental events in which Shh plays a role will be described.

Holoprosencephaly
The importance of Shh signaling in human development became evident by the discovery that mutations in Shh cause Holoprosencephaly (HPE). HPE is a developmental disorder that affects the midline of the face and nervous system. The disorder is characterized by cleft lip and palate, single central incisor, impaired CNS septation, and in severe cases complete cyclopia.5,10 Similar characteristics along with additional phenotypes are observed in mice with a targeted disruption of Shh. The Shh signal is shown to be required for maintenance of the notochord, induction of floorplate and motor neurons, induction of axial skeleton and promotion of distal limb fates.27 Since Shh secreted from the prechordal mesoderm normally signals to inhibit eye formation in the center of eye rudiment, cyclopia results when this signal is defective or missing.1,27 Further confirmation of Shh function in this capacity comes from a plant teratogen, cyclopamine, which causes cyclopia in vertebrate embryos. Cyclopamine, a steroidal alkaloid analogous to cholesterol, acts by inhibiting the ability of target tissues to respond to Shh signaling, probably by antagonizing Smo.28

Left-Right Asymmetry

The first evidence of a role for Shh signaling in vertebrate left-right asymmetry came from experiments in chick embryos. Shh is expressed asymmetrically in the chick node, on the left side during gastrulation. This expression pattern is correlated with normal heart situs (positioning of the heart) later in development.29 Ectopic expression of Shh on the right side causes a randomization of heart situs and induces ectopic expression in the right lateral plate mesoderm of a left-sided marker, nodal.29 Although conservations of left-right asymmetry and of left-sided nodal expression have been described in a variety of vertebrates, there is no evidence of asymmetric hedgehog expression in these other organisms.30 Recent experiments and observations suggest that Shh signaling may still be asymmetric in other vertebrates, even if ligand expression is not. Shh mutant mice exhibit misexpression of left-right markers as well as laterality defects in morphologies of the heart and other organs.31 Interestingly, Shh/Ihh compound mutants have the same phenotypes as Smo mutants. The embryos fail to undergo the normal rightward looping of the heart that is responsible for generating the asymmetry of heart situs.15 In addition, expressions of nodal and other asymmetric left-right markers are absent in Smo mutants.15 Since neither Hedgehog ligands (Shh, Ihh) nor receptors (Smo, Ptc) are asymmetrically expressed,15 it is not clear where the asymmetry of Shh signaling originates. Nonetheless, these results suggest that chick and mouse may utilize similar signaling pathways, such as Shh, to initiate left-right development even though the details of signaling regulation may be different.

(4) Notochord
The notochord is a flexible rod-shaped body found in embryos of all chordates. It is composed of cells derived from the mesoblast and defining the primitive axis of the embryo. In lower vertebrates, it persists throughout life as the main axial support of the body, while in higher vertebrates it is replaced by the vertebral column.

Its appearance synchronizes with that of the neural tube. On the ventral aspect of the neural groove an axial thickening of the endoderm takes place; this thickening assumes the appearance of a furrow (the chordal furrow) the margins of which come into contact, and so convert it into a solid rod of cells (the notochord) which is then separated from the endoderm.

In higher vertebrates, it extends throughout the entire length of the future vertebral column, and reaches as far as the anterior end of the mid-brain, where it ends in a hook-like extremity in the region of the future dorsum sellæ of the sphenoid bone. It lies at first between the neural tube and the endoderm of the yolk-sac, but soon becomes separated from them by the mesoderm, which grows medial-ward and surrounds it. From the mesoderm surrounding the neural tube and notochord, the skull and vertebral column, and the membranes of the brain and medulla spinalis are developed.

@ The QIAexpress® System is based on the remarkable selectivity and affinity of QIAGEN’sexclusive, patented nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography matrices for biomolecules which have been tagged with 6 consecutive histidine residues (6xHis tag)--- affinity chromatography methods.

@ Chimeras can be created from DNA by using conventional cloning techniques, specifically restriction cleavage and DNA ligation.
@ pQE vectors

# Synthetic ribosomal binding site, RBSII, for high translation rates
# 6xHis-tag coding sequence either 5' or 3' to the cloning region
# Two strong transcriptional terminators: t0 from phage lambda (Schwarz et al. 1987), and T1 from the rrnB operon of E. coli, to prevent read-through transcription and ensure stability of the expression construct
# β-lactamase gene (bla) conferring resistance to ampicillin (Sutcliffe 1979) at 100 µg/ml.

@ Invitrogen's TOPO TA Cloning System (TOPO Cloning):
 a fast, efficient method for cloning polymerase chain reaction (PCR) products into a plasmid vector.  To understand TOPO Cloning's design features we must first briefly review the mechanism and products of PCR.
    PCR is a method which essentially isolates a specific segment of DNA by amplification.  PCR uses specific primers which allow Taq DNA polymerase to make copies of only a specific segment. Taq DNA polymerase is used for its ability to withstand the high heat (95C) necessary for the many cycles of PCR.  A unique aspect of Taq DNA polymerase is that it adds a single deoxyadenosine (A) to the 3' ends of PCR products.  The resulting products of PCR are many copies of a specific DNA sequence with 3' A overhangs. 
    TOPO Cloning ligates the PCR product into the pCR 2.1-TOPO plasmid vector (vector) with topoisomerase I.  This can be done quickly and efficiently because of the unique aspects of the vector.  The vector has been engineered to be a linearized plasmid with 3' deoxythymidine (T) overhangs that is activated by being covalently bonded to topoisomerase I.  The 3' A overhangs of the PCR product complement the 3' T overhangs of the vector and allow for fast ligation with the already present topoisomerase I.  The plasmid can then be transformed into competent bacterial cells
    Other useful features of the vector are its ampicillin and kanamycin resistance markers, lacZ reporter gene, T7 promoter, EcoR I sites flanking the PCR insertion site, and the f1 origin of replication.  The ampicillin and kanamycin resistance inserts in the plasmid allow for quick selection of bacterial colonies that take up the vector plasmid during transformationThe lacZ gene in bacteria cause colonies to have a blue color.  If PCR DNA is inserted in the vector it will insert in the middle of the lacZ gene causing the colonies to be white and easily selected.  The T7 promoter region in the vector allows for in vitro RNA transcription/translation by the T7 phage.  If T7 phage infects the bacteria it will make proteins from the DNA sequence of the vector and insert which allows scientists to sequence the subsequent protein and therfore DNA sequence of the insert.  The EcoR I sites on either side of the insert enable the insert to be easily removed by EcoR I restriction enzymes.  The f1 origin of replication is a necessary component of the plasmid and makes single-strand rescue possible. 
    The entire TOPO Cloning processcan be done in only five minutes at room temperature with 95% efficiency.  This is much faster and less complicated than the PRIME PCR Cloner Cloning System we used in lab and most other cloning procedures.  TOPO Cloning system is ideal for scientists that need to quickly clone PCR products into bacteria.

## X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) is a noninducing chromogenic substrate for beta-galactosidase, which hydrolyzes X-Gal forming an intense blue precipitate. X-GaI is most frequently used in conjunction with IPTG in blue/white colony screening to detect recombinants (white) from non-recombinants (blue).    
It is also utilized for selection of beta-galactosidase reporter gene activity in transfection of eucariotic cells and for detection of beta-galactosidase in immunology and histochemistry applications.

@ DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA.

The most popular method for doing this is called the dideoxy method.

DNA is synthesized from four deoxynucleotide triphosphates. Each new nucleotide is added to the 3′ -OH group of the last nucleotide added. 
The dideoxy method gets its name from the critical role played by synthetic nucleotides that lack the -OH at the 3′ carbon atom . A dideoxynucleotide (dideoxynucleotides triphosphate — ddNTP ) can be added to the growing DNA strand but when it is, chain elongation stops because there is no 3′ -OH for the next nucleotide to be attached to. For this reason, the dideoxy method is also called the chain termination method.


The Procedure= 
The DNA to be sequenced is prepared as a single strand.
This template DNA is supplied with
a mixture of all four normal (deoxy) nucleotides in ample quantities dATP
dGTP
dCTP
dTTP
a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
ddATP
ddGTP
ddCTP
ddTTP
DNA polymerase I
@
A. Phenol extraction of DNA samples
Phenol extraction is a common technique used to purify a DNA sample. Typically, an equal volume of TE-saturated phenol is added to an aqueous DNA sample in a microcentrifuge tube. The mixture is vigorously vortexed, and then centrifuged to enact phase separation. The upper, aqueous layer carefully is removed to a new tube, avoiding the phenol interface and then is subjected to two ether extractions to remove residual phenol. An equal volume of water-saturated ether is added to the tube, the mixture is vortexed, and the tube is centrifuged to allow phase separation. The upper, ether layer is removed and discarded, including phenol droplets at the interface. After this extraction is repeated, the DNA is concentrated by ethanol precipitation.

Protocol
1. Add an equal volume of TE-saturated phenol to the DNA sample contained in a 1.5 ml microcentrifuge tube and vortex for 15-30 seconds.
2. Centrifuge the sample for 5 minutes at room temperature to separate the phases.
3. Remove about 90% of the upper, aqueous layer to a clean tube, carefully avoiding proteins at the aqueous:phenol interface. At this stage the aqueous phase can be extracted a second time with an equal volume of 1:1 TE-saturated phenol:chloroform, centrifuged and removed to a clean tube as above but this additional extraction usually is not necessary if care is taken during the first phenol extraction.
4. Add an equal volume of water-saturated ether, vortex briefly, and centrifuge for 3 minutes at room temperature. Remove and discard the upper, ether layer, taking care to remove phenol droplets at the ether:aqueous interface. Repeat the ether extraction.
5. Ethanol precipitate the DNA by adding 2.5-3 volumes of ethanol-acetate, as discussed below.

B. Concentration of DNA by ethanol precipitation
Typically, 2.5 - 3 volumes of an ethanol/acetate solution is added to the DNA sample in a microcentrifuge tube, which is placed in an ice-water bath for at least 10 minutes. Frequently, this precipitation is performed by incubation at -20C overnight. To recover the precipitated DNA, the tube is centrifuged, the supernatant discarded, and the DNA pellet is rinsed with a more dilute ethanol solution. After a second centrifugation, the supernatant again is discarded, and the DNA pellet is dried in a Speedy-Vac.

C. Restriction digestion
Restriction enzyme digestions are performed by incubating double-stranded DNA molecules with an appropriate amount of restriction enzyme, in its respective buffer as recommended by the supplier, and at the optimal temperature for that specific enzyme. The optimal sodium chloride concentration in the reaction varies for different enzymes, and a set of three standard buffers containing three concentrations of sodium chloride are prepared and used when necessary. Typical digestions included a unit of enzyme per microgram of starting DNA, and one enzyme unit usually (depending on the supplier) is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature. These reactions usually are incubated for 1-3 hours, to insure complete digestion, at the optimal temperature for enzyme activity, typically 37degC. See the Appendix for a listing of restriction sites present in the M13 (pUC) MCS and a listing of various restriction enzymes, incubation conditions and cut sites.
D. Agarose gel electrophoresis
Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion, to quickly determine the yield and purity of a DNA isolation or PCR reaction, and to size fractionate DNA molecules, which then could be eluted from the gel. Prior to gel casting, dried agarose is dissolved in buffer by heating and the warm gel solution then is poured into a mold (made by wrapping clear tape around and extending above the edges of an 18 cm X 18 cm glass plate), which is fitted with a well-forming comb. The percentage of agarose in the gel varied. Although 0.7% agarose gels typically are used, in cases where the accurate size fractionation of DNA molecules smaller than 1 kb is required, a 1, 1.5, or 2% agarose gel is prepared, depending on the expected size(s) of the fragment(s). Ethidium bromide is included in the gel matrix to enable fluorescent visualization of the DNA fragments under UV light. Agarose gels are submerged in electrophoresis buffer in a horizontal electrophoresis apparatus. The DNA samples are mixed with gel tracking dye and loaded into the sample wells. Electrophoresis usually is at 150 - 200 mA for 0.5-1 hour at room temperature, depending on the desired separation. When low-melting agarose is used for preparative agarose gels, electrophoresis is at 100-120 mA for 0.5-1 hour, again depending on the desired separation, and a fan is positioned such that the heat generated is rapidly dissipated. Size markers are co-electrophoresed with DNA samples, when appropriate for fragment size determination. Two size markers are used, phi-X 174 cleaved with restriction endonuclease HaeIII to identify fragments between 0.3-2 kb and lambda phage cleaved with restriction endonuclease HindIII to identify fragments between 2-23 kb. After electrophoresis, the gel is placed on a UV light box and a picture of the fluorescent ethidium bromide-stained DNA separation pattern is taken with a Polaroid camera.

E. Elution of DNA fragments from agarose
DNA fragments are eluted from low-melting temperature agarose gels using an unpublished procedure first developed by Dr. Roe. Here, the band of interest is excised with a sterile razor blade, placed in a microcentrifuge tube, frozen at -70degC, and then melted. Then, TE-saturated phenol is added to the melted gel slice, and the mixture again is frozen and then thawed. After this second thawing, the tube is centrifuged and the aqueous layer removed to a new tube. Residual phenol is removed with two ether extractions, and the DNA is concentrated by ethanol precipitation.

F. Kinase end-labeling of DNA
Typical 5'-kinase labeling reactions included the DNA to be labeled, [[gamma]]-32-P-rATP, T4 polynucleotide kinase, and buffer (3). After incubation at 37degC, reactions are heat inactivated by incubation at 80degC. Portions of the reactions are mixed with gel loading dye and loaded into a well of a polyacrylamide gel and electrophoresed. The gel percentage and electrophoresis conditions varied depending on the sizes of the DNA molecules of interest. After electrophoresis, the gel is dried and exposed to x-ray film, as discussed below for radiolabeled DNA sequencing.

G. Bacterial cell maintenance
Four strains of E. coli are used in these studies: JM101 for M13 infection and isolation (4), XL1BMRF' (Stratagene) for M13 or pUC-based DNA transformation (5), and ED8767 for cosmid DNA transformation (6-8). To maintain their respective F' episomes necessary for M13 viral infection (9), JM101 is streaked onto a M9 minimal media (modified from that given in reference (1) plate and XL1BMRF' is streaked onto an LB plate (1) containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid media are inoculated with a smear of several colonies and incubated at 37degC for 8 hours, and those cultures then are transferred into 50 ml of respective liquid media and further incubated 12-16 hours. Glycerol is added to a final concentration of 20%, and the glycerol stock cultures are distributed in 1.3 ml aliquots and frozen at -70degC until use (1).
H. Fragment purification on Sephacryl S-500 spin columns
DNA fragments larger than a few hundred base pairs can be separated from smaller fragments by chromatography on a size exclusion column such as Sephacryl S-500. To simplify this procedure, the following mini-spin column method has been developed.


 

 


@ observational gait analysis:
gross normal  apropulsive knee buckling stiff knee toe-out others
@ gastrocnemius muscle testing
single heel rising test(+/-)   double heel rising test
(+/-)   walk on toe(+/-)
@ functional (disability) assesment
-- grade: 沒有
困難 稍為困難 明顯困難 無法完成
1, 蹲下來再站起來
2, 原地跳10下
3, 平地行走超過30分鐘
4, 爬3層樓的樓梯
5, 小跑步10分鐘
@ symptoms
--- 無 有時 經常 總是
1, 軟腳無力 或 快要跌倒的感覺(giving away)
2, 小腿疼痛
3, 自己或旁人覺得步態異常
4, 下肢麻木感(numbness)
5, 行走或運動耐力下降(endurance decrease)
@ cardiopulmonary test
step test

 



 


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