Frog albumin is expressed in skin and characterized as a novel potent trypsin inhibitor

A novel potent trypsin inhibitor was purified and characterized from frog Bombina maxima skin. A full-length cDNA encoding the protein was obtained from a cDNA library constructed from the skin. Sequence analysis established that the protein actually comprises three conserved albumin domains. B. maxima serum albumin was subsequently purified, and its coding cDNA was further obtained by PCR-based cloning from the frog liver. Only two amino acid variations were found in the albumin sequences from the skin and the serum. However, the skin protein is distinct from the serum protein by binding of a haem b (0.95 mol/mol protein). Different from bovine serum albumin, B. maxima albumin potently inhibited trypsin. It bound tightly with trypsin in a 1: 1 molar ratio. The equilibrium dissociation constants (K-D) obtained for the skin and the serum proteins were 1.92 x 10(-9) M and 1.55 x 10(-9) M, respectively. B. maxima albumin formed a noncovalent complex with trypsin through an exposed loop formed by a disulfide bond (Cys(53)-Cys(62)), which comprises the scissile bond Arg(58)(P-1)-His(59)(P-1'). No inhibitory effects on thrombin, chymotrypsin, elastase, and subtilisin were observed under the assay conditions. Immunohistochemical study showed that B. maxima albumin is widely distributed around the membranes of epithelial layer cells and within the stratum spongiosum of dermis in the skin, suggesting that it plays important roles in skin physiological functions, such as water economy, metabolite exchange, and osmoregulation.

Establishment of a Chinese sturgeon Acipenser sinensis tail-fin cell line and its susceptibility to frog iridovirus

Chinese sturgeon Acipenser sinensis, a cartilaginous ganoid, is a 'living fossil' on a deeply isolated evolutionary branch. A cell line was established from Chinese sturgeon tail-fin tissue (CSTF) . These epithelial CSTF cells grew well in Dulbecco's modified Eagle's medium at 25 degrees C. Karyotypic analysis revealed a normal diploid karyotype with 2n = 264 and large numbers of punctate chromosomes. A strain of frog iridoviruses [Rana grylio virus (RGV)] was used to test the susceptibility of this cell line to infection. Infection was confirmed by cytopathic effect, immunofluorescence and electron-microscope observations, which detected the viral antigens or particles in the cytoplasm of RGV-infected cells. Molecular analysis further suggested that c. 550 bp DNA fragment could be cloned from the RGV-infected CSTF cells' DNA with major capsid protein gene polymerase chain reaction primers. Furthermore, after transfection with pEGFP vector DNA, the CSTF cell line produced significant fluorescent signals indicating its utility in exogenous studies.

猕猴多潜能成体肝前体细胞及猕猴ES 定向分化为胆管上皮细胞的研究

为解决供体器官的不足，以细胞移植为基础的替代疗法已成为治疗不可逆肝 脏疾病新的希望。 肝前体（干）细胞（Hepatic progenitor cellS，HPCs）和 胚胎干细胞（embryoic stem cells, ES）由于其特殊的细胞特性已成为细胞替 代治疗理想的种源细胞。 然而一方面包括人在内的灵长类动物的正常成体肝来 源的HPCs 的分离依然是很困难的；另一方面，ES 细胞来源的肝细胞和胆管细胞 的生成效率依旧很低。因此有必要建立稳定的高效的灵长类动物HPCs 细胞分离 培养体系及ES 细胞的肝细胞或胆管细胞分化体系以满足供体细胞的不足；这种 体系的建立还有利于研究肝细胞生物学如分化机制、自我更新机制等方面的重要 基础问题。 本研究以猕猴为实验模型，研究了正常成体肝来源的猕猴HPCs 分离、纯化 的条件，系统地鉴定了猕猴HPCs 的细胞特性和体内、外分化潜能，并评价了体 内移植效果。 同时以rES 为材料，建立了rES 高效分化为限定性内胚层 （definitive endoderm cells, DE）和胆管上皮细胞的分化体系。主要实验结 果包括：1）： FBS、EGF、HGF 及rat tail collagen (鼠尾胶原)是分离培养正 常成体猕猴来源的肝上皮前体细胞(rhesus monkey liver epithelial progenitor cells, mLEPCs)所必需的，mLEPCs 在此培养体系中至少可以扩增20 代或5 个月以上，并仍然保持原有的细胞特性；mLEPCs 呈现典型的上皮细胞形 态，并表达HPCs 细胞特有的表达模式即同时表达肝细胞和胆管细胞相关基因 （ALB，APOH，CX43，IB4）或蛋白（CK7，CK8，CK18）；在适宜的分化体系下， mLEPCs 可分化为功能性的肝细胞，形成具有胆管上皮细胞的胆管样结构，并能 转分化形成肌肉样细胞、肌样成纤维细胞及少突样细胞；移植入肝损伤的免疫抑 制的小鼠体内后，mLEPCs 能参与受体肝组织的再生，并能分化成ALB 阳性的肝 细胞；体内定位发现mLEPCs 与胆管区的细胞有相似的免疫原性，提示mLEPCs 可能来源于胆管区。2）：rES 在高浓度的acitvin A（100ng/ml）和低浓度的血 清(1%)单层诱导体系下可定向分化得到高比率的限定性内胚层细胞（definitive endoderm cells，DE 细胞）(约80%)； 高比率的DE 细胞的得到还与rES 细胞的接种密度相关；BMP4 和FGF1 可诱导DE 细胞高效向胆管上皮细胞分化（约90%）， 但并不能得到肝细胞；而Notch 信号通路可维持DE 细胞的存活，并决定着DE 细胞向胆管细胞分化，在Notch 信号通路失活的情形下，即使存在BMP4 和FGF1 都不能促使DE 细胞向胆管细胞分化。 本实验首次成功建立了正常猕猴成体肝HPCs 分离培养体系，证实了分离得 到的猕猴肝上皮前体细胞不但具有正常HPCs 的增殖活力和参与受体肝组织的再 生能力，而且还具有三个胚层的分化潜能，这一结果将为以HPCs 为基础的细胞 替代治疗人类肝脏疾病的实现提供了可能，并首次证明了HPCs 也可以像某些少 数成体干细胞一样具有三个胚层得分化潜能。 此外，本研究建立了rES 高效定 向分化为DE 细胞和胆管细胞的分化体系，这一方法的建立将促进灵长类动物的 DE 细胞的发育机制研究，同时也可为高比率的内胚层功能细胞（如胰岛细胞、 肝细胞、肺细胞）的获得提供丰富的种源细胞和平台。

Diagnosis of colorectal cancer using Raman spectroscopy of laser-trapped single living epithelial cells

A single-cell diagnostic technique for epithelial cancers is developed by utilizing laser trapping and Raman spectroscopy to differentiate cancerous and normal epithelial cells. Single-cell suspensions were prepared from surgically removed human colorectal tissues following standard primary culture protocols and examined in a near-infrared laser-trapping Raman spectroscopy system, where living epithelial cells were investigated one by one. A diagnostic model was built on the spectral data obtained from 8 patients and validated by the data from 2 new patients. Our technique has potential applications from epithelial cancer diagnosis to the study of cell dynamics of carcinogenesis. (c) 2006 Optical Society of America.

Cultured gill epithelial cells from tilapia (Oreochromis niloticus): a new in vitro assay for toxicants

A culture gill epithelium from seawater-adapted tilapia (Oreochromis niloticus) was developed for testing PAHs and dioxin-like contaminants in seawater. The epithelia consists two to three layers of epithelial cells incorporating both pavement cells and mitochondria-rich cells (MRCs). Polarity and a stable transepithelial resistance (TER) were maintained. and closely resembled those in fish gills in vivo. The tightness (integrity) of the epithelia remained unchanged upon exposure to benzo[a]pyrene (B[a]P). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3',4,4',5-pentachlorobiphenyl (PCB#126), while a concentration-dependent response of EROD activity in the epithelia was induced within 18-24 h when the apical side was exposed to these toxicants. The 24 h EC50 of EROD activity was 2.77 x 10(-7) M for PCB#126, 1.85 x 10(-7) M for B[a]P and 7.38 x 10(-10) M for TCDD. showing: that the preparation was not only sensitive to PAHs and dioxin-like compounds, but also able to produce inductive potency of AhR agonists that generally agreed with those derived from other established in vitro and in vivo systems. The results suggest, that the cultured gill epithelia from seawater-adapted tilapia may serve as a simple. rapid and cost-effective tool for assessing exposure and potential effects of toxicants in marine waters. (C) 2004 Elsevier B.V. All rights reserved.

Staphylococcus aureus lipoproteins trigger human corneal epithelial innate response through toll-like receptor-2

Bacterial lipoproteins (LP) are a family of cell wall components found in a wide variety of bacteria. In this study, we characterized the response of HUCL, a telomerase-immortalized human corneal epithelial cell (HCEC) line, to LP isolated from Staphylococcus (S) aureus. S. aureus LP (saLP) prepared by Triton X-114 extraction stimulated the activation of NF-kappa B, JNK, and P38 signaling pathways in HUCL cells. The extracts failed to stimulate NF-kappa B activation in HUCL cells after lipoprotein lipase treatment and in cell lines expressing TLR4 or TLR9, but not TLR2, indicating lipoprotein nature of the extracts. saLP induced the up-regulation of a variety of inflammatory cytokines and chemokines (IL-6, IL-8, ICAM-1). antimicrobial molecules (hBD-2, LL-37, and iNOS), and homeostasis genes (Mn-SOD) at both the mRNA level and protein level. Similar inflammatory response to saLP was also observed in primarily cultured HCECs using the production of IL-6 as readout. Moreover, TLR2 neutralizing antibody blocked the saLP-induced secretion of IL-6, IL-8 and hBD2 in HUCL cells. Our findings suggest that saLP activates TLR2 and triggers innate immune response in the cornea to S. aureus infection via production of proinflammatory cytokines and defense molecules. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of subretinal implant materials on the viability, apoptosis and barrier function of cultured RPE cells

Background: Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells.Methods: Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function.Results: The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility.Conclusions: The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.