Comparison of Long‐Term Survival of Pigmented Epidermal Reconstructs Cultured In Vitro vs. Xenografted on Nude Mice

Epidermal reconstructs incorporating pigment cells have been used in vitro over the last decade to study the physiology of the epidermal melanin unit. However, the major limitation of this technology is the duration of the assays, which need to be completed within 2–3 weeks to obviate the problem of epidermal senescence and excessive terminal differentiation. This becomes a major problem for studying long‐term biological phenomena in photoprotection and epidermal skin cancers. We report here a simplified surgical technique in immunotolerant mice allowing long‐term studies. The creation of a vascularized mouse skin flap is the key point of the surgical procedure. Long‐term pigmentation of the xenografts seemed macroscopically successful, but surprisingly microscopy at 11 and 16 weeks postgrafting showed mostly dermal pigment aggregates and rare Melan‐A positive dermal and epidermal pigment cells. In the same reconstructs maintained in vitro, dermal pigment and dermal pigment cells were never noted. It could be speculated that in our model, the colonization of the xenografted dead human dermis by murine cells influences melanocyte survival.     

14种木犀榄族植物叶表皮微形态的研究

利用光学显微镜和扫描电子显微镜对木犀榄族（Oleeae）5属14种植物的叶表皮微形态特征进行了观察,结果表明：（1）叶表皮细胞形状有无规则形和多边形两种;下表皮腺点的数目远远大于上表皮,流苏树上表皮细胞没有腺点;叶下表皮有气孔器,形状为圆形、椭圆形;气孔器均为不规则型;气孔器外围角质层有放射状、条状、环状、颗粒状等多种类型;（2）气孔器和叶表皮细胞许多特征具有明显的种间差异,可以作为种间鉴定的重要依据;（3）叶表皮微形态结构上的许多共同特征,表明木犀榄族植物是单独一自然类群。上述特征可为木犀榄族植物属及物种的划分与鉴定、系统演化关系的探讨等提供参考。     

Gene Expression Profile of Glioblastoma Peritumoral Tissue: An Ex Vivo Study

The gene expression pattern of glioblastoma (GBM) is well documented but the expression profile of brain adjacent to tumor is not yet analysed. This may help to understand the oncogenic pathway of GBM development. We have established the genome-wide expression profiles of samples isolated from GBM tumor mass, white matter adjacent to tumor (apparently free of tumor cells), and white matter controls by using the Affymetrix HG-U133 arrays. Array-CGH (aCGH) was also performed to detect genomic alterations. Among genes dysregulated in peritumoral white matter, 15 were over-expressed, while 42 were down-regulated when compared to white matter controls. A similar expression profile was detected in GBM cells. Growth, proliferation and cell motility/adhesion-associated genes were up-regulated while genes involved in neurogenesis were down-regulated. Furthermore, several tumor suppressor genes along with the KLRC1 (a member of natural killer receptor) were also down-regulated in the peritumoral brain tissue. Several mosaic genomic lesions were detected by aCGH, mostly in tumor samples and several GBM-associated mosaic genomic lesions were also present in the peritumoral brain tissue, with a similar mosaicism pattern. Our data could be explained by a dilution of genes expressed from tumor cells infiltrating the peritumour tissue. Alternatively, these findings could be substained by a relevant amount of “apparently normal” cells presenting a gene profile compatible with a precancerous state or even “quiescent” cancer cells. Otherwise, the recurrent tumor may arise from both infiltrating tumor cells and from an interaction and recruitment of apparently normal cells in the peritumor tissue by infiltrating tumor cells.     

天南星科叶表皮研究

利用光学显微镜对天南星科18属27种及菖蒲科1属1种植物的叶表皮微形态进行观察,同时用扫描电镜对具代表性的14种植物作了研究,结果显示：天南星科气孔类型变异较大,有不规则型,辐射型,平列型,胞环型及平列型和胞环型间的过渡类型,副卫细胞数目0－12个;表皮细胞长宽近相等,平周壁具条纹或否,垂周壁平直,弧形或波浪形,虽然气孔类型对天南星科分类上的意义不大,但与表皮细胞垂周壁形状,副卫细胞角质层纹饰等特征相结合对种间分类有一定意义,天南星科与菖蒲科叶表皮微形态明显不同,从而支持菖蒲属从天南星科中分出另立为科的观点。     

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.     

中国卫矛属植物叶表皮微形态特征及其分类学意义

利用光学显微镜和扫描电子显微镜对中国卫矛属(Euonymus L.)24种以及沟瓣属（Glyptopetalum Thw.）3种植物的叶表皮微形态特征进行了观察研究,测量并统计了气孔器长宽比、密度和气孔指数。结果显示,两属植物叶表皮微形态特征基本一致：叶上、下表皮细胞形状多为多边形,垂周壁平直-弓形;气孔器通常仅分布在叶下表皮;均具有无规则型气孔器,部分种还具环列型气孔器。扫描电镜下角质膜纹饰、表皮细胞界限及气孔特征在一些种间存在差异,可以作为区分部分近缘种类的参考依据。     

中国西南地区独活属植物叶表皮微形态特征及其系统学意义

利用光学显微镜和扫描电镜对中国西南地区独活属16种1变种植物(分隶于中国独活属的全部4个组)的叶表皮微形态进行了观察,测量并统计气孔器大小、密度和气孔指数,并用统计学方法对远轴面气孔器长轴进行显著差异性分析.结果显示:无规则型气孔器普遍存在于所有研究类群的叶远轴面及个别类群叶近轴面,气孔器的分布和密度具有种间特异性.表皮毛普遍存在于远轴面及大部分类群近轴面,长短和覆盖密度因种而异.近轴面表皮细胞为多边形或者不规则形,垂周壁平直、浅波状或波状;远轴面表皮细胞形态多不规则形,表皮细胞垂周壁浅波状或波状.在扫描电镜下,叶表皮气孔器外拱盖内缘为近平滑、浅波状或波状;角质膜条纹状,有的附有颗粒状、鳞片状蜡质等结构.气孔器外拱盖形态以及蜡质类型是稳定的鉴别特征.研究表明独活属植物叶表皮特征存在较大的种间差异,对独活属的系统分类及进化研究具有重要意义,文中对叶表皮特征在独活属植物分类处理中的应用及系统进化问题等进行了讨论,并建立了以叶表皮微形态特征为依据的分类检索表.     

An Ex Vivo Method for Evaluating Prostaglandin Synthetase Activity in Cortical Slices of Mouse Brain

The release of prostaglandin E2 (PGE2) from cortical slices of mice into incubation medium is followed for 3 h and compared to PGE2 levels in the corresponding slice. Immediately after decapitation, the rate of PGE2 released into the incubation medium is elevated and a steady low rate of spontaneous release is gained within 1-2 h of incubation. PGE2 synthesis and release is blocked in a dose-dependent manner by either indomethacin (3 X 10(-6) -3 X 10(-4) M) or flufenamic acid (2.6 X 10(-6) M) either when added in vitro or administered in vivo. Full recovery of PGE2 synthesis is reached after 3 h incubation of slices following in vivo administration of indomethacin. In vivo administration of flufenamic acid results in prolonged inhibition of PGE2 released in vitro. The inhibition of PGE2 released by indomethacin is also correlated with the slice PGE2 content. Administration of lipopolysaccharide (LPS), a known activator of phospholipase A2, results in a fivefold increase in PGE2 and a twofold increase in 6-keto-PGF1 alpha released into the medium. The release of thromboxane B2 is not affected by LPS.     

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.     

紫外线对甲型肝炎病毒灭活机理的研究

自1979年Provost体外培养甲型肝炎病毒（HAV）首获成功以来［1］,有关HAV对多种理化因子抗力的研究不少【’1,但对其具体作用机制,迄今尚未见诸报道。为此,本研究通过观察UV照射对HAVRNA链与衣壳蛋白完整性等的影响,探讨UV对HAV的灭活机理,为灭活HAV的实际应用提供理论基础。材料与方法1病毒与细胞受试毒株为HAVHM－175株,其培养细胞为非洲绿猴肾细胞MEK株,生长液为Engle”s液。生长与维持培养温度分别为37℃和35℃,2病毒灭活试验将纯化HAV悬液均匀涂布于细胞培养板孔内,室温干燥,置照射强度为300pw八mZ紫外灯下6…     

中国红豆属植物的叶表皮形态学

用光学显微镜和扫描电子显微镜观察了红豆属(Ormosia)35种的叶表皮形态,这些植物的叶上下表皮细胞为不规则形或多边形,垂周壁有平直、弓形、浅波状或深波状,表皮细胞形状和垂周壁在种间有一些差异。气孔仅存在于下表皮,形状为椭圆形或卵形,以平列型为主;此外还有其他类型的气孔,如长脐红豆、海南红豆等的气孔为不规则形、不等型;少数种类如亮毛红豆、茸荚红豆等的气孔不明显。气孔外拱盖光滑,呈浅波状、波状或不明显,气孔内拱盖光滑、近光滑、浅波状或不明显;蜡被近光滑、颗粒状、鳞片状、条纹状或结网状;多数种具毛被。叶表皮与气孔器特征可作为区分种、变种或亚种的依据。叶表皮微观形态特征支持红豆属成为一个自然类群。     

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.     

Human Dupuytren's Ex Vivo Culture for the Study of Myofibroblasts and Extracellular Matrix Interactions

Organ fibrosis or “scarring” is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic.To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first “organ” culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.     

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.     

Epidermal differentiation in the developing scales of embryos of the Australian scincid lizard Lampropholis guichenoti

Formation of the first epidermal layers in the embryonic scales of the lizard Lampropholis guichenoti was studied by optical and electron microscopy. Morphogenesis of embryonic scales is similar to the general process in lizards, with well‐developed overlapping scales being differentiated before hatching. The narrow outer peridermis is torn and partially lost during scale morphogenesis. A second layer, probably homologous to the inner peridermis of other lizard species, but specialized to produce lipid‐like material, develops beneath the outer peridermis. Two or three lipogenic layers of this type develop in the forming outer surface of scales near to the hinge region. These layers form a structure here termed “sebaceous‐like secretory cells.” These cells secrete lipid‐like material into the interscale space so that the whole epidermis is eventually coated with it. This lipid‐like material may help to reduce friction and to reduce accumulation of dirt between adjacent extremely overlapping scales. At the end of their differentiation, the modified inner periderm turns into extremely thin cornified cells. The layer beneath the inner peridermis is granulated due to the accumulation of keratohyalin‐like granules, and forms a shedding complex with the oberhautchen, which develops beneath. Typically tilted spinulae of the oberhautchen are formed by the aggregation of tonofilaments into characteristically pointed cytoplasmic outgrowths. Initially, there is little accumulation of β‐keratin packets in these cells. During differentiation, the oberhautchen layer merges with cells of the β‐keratin layer produced underneath, so that a typical syncytial β‐keratin layer is eventually formed before hatching. Between one‐fourth distal and the scale tip, the dermis under epidermal cells is scarce or absent so that the mature scale tip is made of a solid rod of β‐keratinized cells. At the time of hatching, differentiation of a mesos layer is well advanced, and the epidermal histology of scales corresponds to Stage 5 of an adult shedding cycle. The present study confirms that the embryonic sequence of epidermal stratification observed in other species is basically maintained in L. guichenoti. J. Morphol. 241:139–152, 1999. © 1999 Wiley‐Liss, Inc.     

An analysis of the repair processes in ultraviolet-irradiated Micrococcus luteus using purified ultraviolet-endonuclease

The measurement of the frequency of endonucleolytic incisions in ultraviolet-irradiated DNA serves as the test for the presence of pyrimidine dimers. In accordance with this approach, the lysates of three Micrococcus luteus strains containing radioactively labeled chromosomes were treated with purified M. luteus ultraviolet-endonuclease to trace segregation of dimers amongst parental and newly synthesized DNA and their removal during postreplication and excision DNA repair. A considerable proportion of the dimers in all strains tested proved to be insensitive to the action of exogenous incising enzyme. The use of chloramphenicol as an inhibitor of postirradiation protein synthesis in combination with ultraviolet-endonuclease treatment of DNA allowed to reveal at least two alternative pathways of postreplication repair: constitutively active recombinational pathway and inducible nonrecombinational one.     

国产画眉草亚族叶表皮微形态特征及其在属间关系上的意义

通过国产画眉草亚族叶片表皮的解剖观察,结合外部形态,对该亚族内6个属的属间关系进行了分析。结果表明：羽穗草属应是国产画眉草亚族中最原始的类群,最高级的类群仍数细画眉草属,而其余4属即画眉草属、弯穗草属、尖稃草属和镰稃草属的演化水平居于两者之间;画眉草属和弯穗草属可能直接起源于原始的羽穗草属,而较高级的尖稃草属和镰稃草属又可能直接起源于较原始的画眉草属,并在镰稃草属的基础上进而派生了最进化的细画眉草属。整个研究结果既弥补了前人演化理论的不足,又为今后族进化的全面探讨提供了参考。     

Study on the synergetic degradation of chitosan with ultraviolet light and hydrogen peroxide

Chitosan was effectively degraded by hydrogen peroxide under irradiation with ultraviolet light. The existence of a synergetic effect on the degradation was demonstrated by means of viscometry. In addition, the optimal conditions of degradation were determined on the basis of orthogonal tests. The structure of the degraded product was characterized by Fourier-transform infrared spectra (FTIR) analysis and diffuse reflectance spectra (DRS) analysis. The mechanism of the degradation of chitosan was correlated with cleavage of the glycosidic bond.     

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale. There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation. However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons. Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms. Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation. We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons. This approach is likely to have broad utility in the field of neuronal endocytic trafficking.     

Recovery of extracellular matrix components by enalapril maleate during the repair process of ultraviolet B-induced wrinkles in mouse skin

The renin–angiotensin system is known to be involved in skin remodeling and inflammation. Previously, we reported that ultraviolet B (UVB) irradiation enhanced angiotensin-converting enzyme (ACE) expression and angiotensin II levels in hairless mouse skin, and an ACE inhibitor, enalapril maleate (EM), accelerated repair of UVB-induced wrinkles. In this study, we analyzed gene expression profiles by DNA microarray and protein distribution patterns using an immunofluorescence method to clarify the process of EM-accelerated wrinkle repair in UVB-irradiated hairless mouse skin. In the microarray analysis, we detected EM-induced up-regulation of various extracellular matrix (ECM)-related genes in the UVB-irradiated skin. In the immunofluorescence, we confirmed that type I collagen α1 chain, fibrillin 1, elastin and dystroglycan 1 in the skin decreased after repeated UVB irradiation but staining for these proteins was improved by EM treatment. In addition, ADAMTS2 and MMP-14 also increased in the EM-treated skin. Although the relationship between these molecules and wrinkle formation is not clear yet, our present data suggest that the molecules are involved in the repair of UVB-induced wrinkles.