Photodynamic therapy on keloid fibroblasts in tissue-engineered keratinocyte-fibroblast co-culture

BACKGROUND AND OBJECTIVES: Keloids are disfiguring, proliferative scars that are a pathologic response to cutaneous injury. An organotypic tissue culture system (the Raft model 1-10) was used to investigate the feasibility of using photodynamic therapy (PDT) as an adjunctive therapy to treat keloids following surgical excision. The Raft co-culture system mimics skin by layering keratinocytes on top of fibroblasts embedded in a collagen matrix. PDT uses drugs that produce singlet oxygen in situ when irradiated by light, and may lead to a number of effects in living tissues varying from the modulation of growth to apoptosis. PDT is already used to treat several benign and malignant diseases in organs such as the skin, retina, and esophagus. STUDY DESIGN/MATERIALS AND METHODS: Normal adult, neonatal, and keloid fibroblasts and keratinocytes were isolated from skin obtained from patients undergoing elective procedures and used to construct the Rafts. Mature Rafts (after 4 days) were incubated with 5-amino levulinic acid (5-ALA), a photosensitizer, for 3 hours and were laser-irradiated (635 nm) for total energy delivery of 5 J/cm2, 10 J/cm2, or 20 J/cm2. Rafts were examined 24 hours and 14 days later. Cell viability was determined using confocal imaging combined with live-dead fluorescent dyes. Multi-photon microscope (MPM) imaged collagen structure and density. As Rafts contract over time, surface area was measured using optical micrometry daily. RESULTS: At 10 and 20 J/cm2, near-total cell death was observed in all constructs, while at 5 J/cm2 cell viability was comparable to controls. Cell viability in keloid and neonatal Rafts was greater than that observed in normal adult Rafts. Treated Rafts contracted less over the 14-day period compared to controls. Contraction and collagen density were greatest in keloid and neonatal Rafts. CONCLUSIONS: A PDT dosimetry range was established, which reduces tissue contraction and collagen density while minimizing injury to fibroblasts.     

MT4-(MMP17) and MT6-MMP (MMP25), A unique set of membrane-anchored matrix metalloproteinases: properties and expression in cancer

The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs.     

The role of lipid rafts in cell entry of human metapneumovirus

Human metapneumovirus (hMPV) is a crucial pathogen in children. A cell entry is the first step for infection. Our previous study indicated that there was an endocytosis pathway for hMPV cell entry. Lipid raft is a specific structure at the cell surface and it has been demonstrated to play an important role in endocytosis process of many viruses. In this study, we investigated whether and how lipid raft can take part in the hMPV entry. The confocal microscope was used to detect colocalization of hMPV and lipid raft marker. We demonstrated that colocalizations were increased along with the viral infection and hMPV particles transferred to the perinuclear region with lipid raft. When specific lipid raft inhibitors: methyl-β cyclodextrin and nystatin were used, hMPV cell entry was inhibited and viral titer decreased dramatically. With the replenishment of exogenous cholesterol, hMPV recovered quickly. These data suggest that lipid raft plays an important role in hMPV endocytosis and maybe one of the pathways for hMPV cell entry.     

Cannabidiol-induced apoptosis in murine microglial cells through lipid raft

Cannabidiol (CBD), the major nonpsychotropic phytocannabinoid, induces apoptosis in both immortalized and primary lymphocytes and monocytes. However, contrasting effects of CBD on the apoptosis between normal and immortalized glial cells have been reported. This study investigated the proapoptotic effect of CBD on primary microglial cells. Treatment of murine primary microglial cultures with CBD resulted in a time- and concentration-dependent induction of apoptosis, as shown by increase in hypodiploid cells and DNA strand breaks, and marked activation of both caspase-8 and -9. Mechanistic studies revealed that antioxidants, including N-acetyl-L-cysteine and glutathione, the G protein-coupled receptor 55 agonist abnormal-CBD and specific antagonists for vanilloid, and CB1 and CB2 cannabinoid receptors did not counteract the apoptosis induced by CBD. In contrast, methyl-β-cyclodextrin (MCD), a lipid raft disruptor, potently attenuated CBD-induced microglial apoptosis and caspase activation. Furthermore, CBD induced lipid raft coalescence and augmented the expression of GM1 ganglioside and caveolin-1, all of which were attenuated by MCD. Taken together, these results suggest that CBD induces a marked proapoptotic effect in primary microglia through lipid raft coalescence and elevated expression of GM1 ganglioside and caveolin-1.     

液质联用蛋白组学技术分析脑组织脂筏样品的属性*

目的验证蔗糖密度梯度超速离心法提取脑组织脂筏的有效性。方法用蔗糖密度梯度超速离心法提取小鼠脑组织脂筏,采用免疫印迹法、双酶胆固醇检测法结合光散射度分析样品的脂筏属性,采用液质联用蛋白组学技术和生物信息学手段,对脂筏中的蛋白质细胞定位进行分析。结果超速离心法提取的脑组织脂筏具有典型的高散色度、高胆固醇和高表达Flotillin-1的脂筏特性;液质联用蛋白组学分析从脂筏样本中鉴定出647种蛋白质,这些蛋白质细胞定位大多是细胞膜、内质网、细胞骨架和细胞浆等常见的脂筏蛋白来源,这种脂筏样品是含有杂质的混合物,鉴定出的647种总蛋白中,有21%是细胞核、线粒体和核糖体等非脂筏来源蛋白。结论超速离心法是提取脑组织脂筏的有效方法,但应用中要考虑杂蛋白的影响。     

脂筏募集蛋白分析针刺对阿尔茨海默病小鼠跨膜细胞信号途径的影响

目的揭示针刺治疗阿尔茨海默病（AIzheimers disease,AD）中参与神经元功能调控的跨膜细胞信号途径。方法以SAMP8小鼠为AD动物模型,用液质联用蛋白质组学方法检测益气调血扶本培元针法对早老化痴呆小鼠SAMP8海马脂筏募集的跨膜细胞信号蛋白数量和种类的影响。结果与对照组比较,针刺组小鼠SAMP8海马脂筏募集的细胞信号类蛋白质数量增多39种,其中,离子通道蛋白14种,G蛋白8种,跨膜信号受体8种,激酶9种,共涉及包括G蛋白偶联受体信号、酶耦联受体信号和离子通道介导3类主要的细胞信号途径;与非穴组比较,针刺组海马脂筏募集的激酶类信号蛋白数量显著增加,增加的激酶从功能上看,以调节神经元细胞骨架相关的突触功能和神经递质分泌为主。结论针刺治疗AD的细胞生物学机制,可能是通过多种跨膜细胞信号改善神经元突触的功能和促进神经递质分泌,达到改善AD患者大脑认知功能的效果。     

Lipid raft: A floating island of death or survival

Lipid rafts are microdomains of the plasma membrane enriched in cholesterol and sphingolipids, and play an important role in the initiation of many pharmacological agent-induced signaling pathways and toxicological effects. The structure of lipid rafts is dynamic, resulting in an ever-changing content of both lipids and proteins. Cholesterol, as a major component of lipid rafts, is critical for the formation and configuration of lipid raft microdomains, which provide signaling platforms capable of activating both pro-apoptotic and anti-apoptotic signaling pathways. A change of cholesterol level can result in lipid raft disruption and activate or deactivate raft-associated proteins, such as death receptor proteins, protein kinases, and calcium channels. Several anti-cancer drugs are able to suppress growth and induce apoptosis of tumor cells through alteration of lipid raft contents via disrupting lipid raft integrity.     

Host sphingolipid biosynthesis is a promising therapeutic target for the inhibition of hepatitis B virus replication

Serine palmitoyltransferase (SPT) catalyzes the first step in the sphingolipid biosynthetic pathway. Myriocin inhibits SPT and was shown to suppress the replication of hepatitis C virus (HCV) in vitro and in vivo. However, its effect on hepatitis B virus (HBV) replication is unknown. In this study, the HBV DNA levels in HuH7 cell culture supernatants were lowered successfully by using myriocin and it was found that the 50% inhibitory concentration of myriocin is approximately 5 μM. Myriocin and/or pegylated interferon (PEG-IFN) were also administered to chimeric mice for 2 weeks and the effects of these compounds on HBV DNA levels were determined. Myriocin alone did not reduce effectively the HBV DNA levels, whereas PEG-IFN alone reduced the DNA levels to 1/10th of the control levels. The combination of myriocin with PEG-IFN reduced the HBV levels to about 1/1,000 th of the control levels and induced a 1.0 log reduction in the levels of the HBV surface antigen and core protein. This latter effect was not observed in the other treatment groups. In conclusion, the combination of myriocin with PEG-IFN represses synergistically HBV replication in vivo without inducing hepatotoxicity.     

Biochemical interaction of an actin‐capping protein,CapZ, with NAP‐22

NAP‐22 is a neuronal protein localized in the presynaptic membrane and synaptic vesicles and recovered in a Triton‐insoluble low‐density microdomain fraction after biochemical fractionation of the synaptic plasma membrane. NAP‐22 organizes membrane microdomains through binding to membrane lipids such as cholesterol, phosphatidylethanolamine, and phosphatidylinositol 4,5‐bisphosphate. In this study, NAP‐22‐binding proteins were screened through the pull‐down assay using brain‐derived NAP‐22 bound to Sepharose 4B. An actin‐capping protein, CapZ, was identified in the precipitate through mass spectrometry and Western blotting. CapZ was then expressed in E. coli and the purified protein‐bound NAP‐22 directly. Because bacterially expressed NAP‐22 bound CapZ, it was determined that the N‐terminal myristoyl moiety of NAP‐22 is not necessary for the binding. The binding of NAP‐22 showed no effect on the actin nucleation activity of CapZ measured with centrifugation and viscometric assays. Hence, the CapZ–NAP‐22 complex could work as the nucleation site of actin polymerization or as the actin filament‐anchoring site on the membrane microdomain. © 2009 Wiley‐Liss, Inc.     

The α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins,a posttranslational modification essential for function

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α₂δ subunit; both of these subunits influence the trafficking and properties of Ca_V1 and Ca_V2 channels. The α₂δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α₂δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α₂δ subunits studied, α₂δ-1, α₂δ-2, and α₂δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α₂δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α₂δ subunits. We also show that GPI anchoring of α₂δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α₂δ subunits are coexpressed. In conclusion, this study redefines our understanding of α₂δ subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.