Lysophosphatidic acid induces cell migration through the selective activation of Akt1

Akt plays pivotal roles in many physiological responses including growth, proliferation, survival, metabolism, and migration. In the current studies, we have evaluated the isoform-specific role of akt in lysophosphatidic acid (LPA)-induced cell migration. Ascites from ovarian cancer patients (AOCP) induced mouse embryo fibroblast (MEF) cell migration in a dose-dependent manner. On the other hand, ascites from liver cirrhosis patients (ALCP) did not induce MEF cell migration. AOCP-induced MEF cell migration was completely blocked by pre-treatment of cells with LPA receptor antagonist, Ki16425. Both LPA- and AOCP-induced MEF cell migration was completely attenuated by PI3K inhibitor, LY294002. Furthermore, cells lacking Akt1 displayed defect in LPA-induced cell migration. Re-expression of Akt1 in DKO (Akt1-/-Akt2-/-) cells restored LPA-induced cell migration, whereas re-expression of Akt2 in DKO cells could not restore the LPA-induced cell migration. Finally, Akt1 was selectively phosphorylated by LPA and AOCP stimulation. These results suggest that LPA is a major factor responsible for AOCP-induced cell migration and signaling specificity of Akt1 may dictate LPA-induced cell migration.     

Tie2 is tied at the cell-cell contacts and to extracellular matrix by Angiopoietin-1

Angiopoietin-1 (Ang1) binds to and activates Tie2 receptor tyrosine kinase. Ang1-Tie2 signal has been proposed to exhibit two opposite roles in the controlling blood vessels. One is vascular stabilization and the other is vascular angiogenesis. There has been no answer to the question as to how Tie2 induces two opposite responses to the same ligand. Our group and Dr. Alitalo''s group have demonstrated that trans-associated Tie2 at cell-cell contacts and extracellular matrix (ECM)-anchored Tie2 play distinct roles in the endothelial cells. The complex formation depends on the presence or absence of cell-cell adhesion. Here, we review how Ang1-Tie2 signal regulates vascular maintenance and angiogenesis. We further point to the unanswered questions that must be clarified to extend our knowledge of vascular biology and to progress basic knowledge to the treatment of the diseases in which Ang1-Tie2-mediated signal is central.     

A concept for miniaturized 3-D cell culture using an extracellular matrix gel

This paper presents a novel method to embed, anchor, and cultivate cells in a controlled 3-D flow-through microenvironment. This is realized using an etched silicon pillar flow chamber filled with extracellular matrix (ECM) gel mixed with cells. At 4 degrees C, while in liquid form, ECM gel is mixed with cells and injected into the chamber. Raising the temperature to 37 degrees C results in a gel, with cells embedded. The silicon pillars both stabilize and increase the surface to volume ratio of the gel. During polymerization the gel shrinks, thus creating channels, which enables perfusion through the chip. The pillars increase the mechanical stability of the gel permitting high surface flow rates without surface modifications. Within the structure cells were still viable and proliferating after 6 days of cultivation. Our method thus makes it possible to perform medium- to long-term cultivation of cells in a controlled 3-D environment. This concept opens possibilities to perform studies of cells in a more physiological environment compared to traditional 2-D cultures on flat substrates.     

Molecular design of three-dimensional artificial extracellular matrix: Photosensitive polymers containing cell adhesive peptide

A photodimerizable monomer, methacrylic acid-(7-coumaroxy) ethyl ester, was synthesized and was copolymerized with a hydrophilic monomer (N,N-dimethylacrylamide) to obtain a water-soluble photosensitive polymer. Irradiation of the copolymer film and aqueous solution with a high-pressure mercury lamp resulted in highly hydrated gel. The gel yield was enhanced with the content of the photodimerizable group in the copolymer and the irradiation time. The degree of swelling of the gels decreased concomitantly. Incorporation of the well-known cell adhesive peptidyl ligand Arg-Gly-Asp-Ser(RGDS) into photosensitive copolymers attained a biologically active hydrophilic gel matrix upon UV light irradiation. Irradiation of a buffer solution of the latter copolymer premixed with smooth muscle cells entrapped the cells throughout the gel matrix. This indicates that the designed polymer and the resulting cell-incorporated hydrogel are biomimic to an extracellular matrix and to the media of the vascular wall, respectively. © 1993 John Wiley & Sons, Inc.     

Hybrid Scaffolds Built From PET and Collagen as a Model For Vascular Graft Architecture

A hybrid material with excellent mechanical and biological properties is produced by electrospinning a co‐solution of PET and collagen. The fibers are mapped using SEM, confocal Raman microscopy and collagenase digestion assays. Fibers of different compositions and morphologies are intermingled within the same membrane, resulting in a heterogeneous scaffold. The collagen distribution and exposure are found to depend on the PET/collagen ratio. The materials are chemically and mechanically characterized and biologically tested with fibroblasts (3T3‐L1) and a HUVEC culture in vitro. All of the hybrid scaffolds show better cell attachment and proliferation than PET. These materials are potential candidates to be used as vascular grafts.

     

Inhibitory effect of capsaicin on B16-F10 melanoma cell migration via the phosphatidylinositol 3-kinase/Akt/Rac1 signal pathway

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), the major pungent ingredient of red pepper, has been reported to possess anti-carcinogenic and anti-mutagenic activities. In this study, the anti-migration activity of capsaicin on highly metastatic B16-F10 melanoma cells was investigated. Capsaicin significantly inhibited the migration of melanoma cells without showing obvious cellular cytotoxicity at low doses. This effect correlated with the down-regulation of phosphatidylinositol 3-kinase (PI3-K) and its downstream target, Akt. Although B16-F10 cell migration was increased by the PI3-K activator through the activation of Akt, these PI3-K activator-induced phenomena were attenuated by capsaicin. Moreover, capsaicin was found to significantly inhibit Rac1 activity in a pull-down assay. These results demonstrate that capsaicin inhibits the migration of B16-F10 cells through the inhibition of the PI3-K/Akt/Rac1 signal pathway. The present investigation suggests that capsaicin targets PI3-K/Akt/ Rac1-mediated cellular events in B16-F10 melanoma cells. Consequently, capsaicin administration should be considered an effective approach for the suppression of invasion and metastasis in malignant melanoma chemotherapy.     

Recent progress in plant cell culture

Conclusions Commercial application of plant cell cultures for the production of useful metabolites on a large scale has not yet been realized. However, some recently published papers and patents seem very attractive for industrial use because their results and techniques are apparently applicable to a large-scale production of metabolites. The author believes that commercial success using plant tissue cultures will be realized in the near future with advanced techniques if product selection is determined by economic and political principles. In 1982, the 5th International Congress of Plant Tissue and Cell Culture will be held in Japan and this undoubtedly will encourage research activity in our country.     

Vanadium determination in chloride matrices using ICP-MS: finding the optimum collision/reaction cell parameters for suppressing polyatomic interferences

Efficiencies of He/NH₃ and He/H₂ collision gases were compared in a conventional type of hexapole cell of an inductively coupled plasma mass spectrometer (ICP-MS). The optimum conditions [hexapole and quadrupole bias voltage (V_H and V_Q) and collision/reaction gas flow rates] were tested for vanadium determination (⁵¹V) in chloride matrices. When the He/H₂ mixture was used, the optimum values of V_H and V_Q were −10.0 and −8.0 V, respectively. This set-up corresponds to the kinetic energy discrimination effect. When the He/NH₃ mixture was used, the optimum values of V_H and V_Q were +10.0 and −7.0 V, respectively. Positive V_H values correspond to the ion kinetic energy effect, which allows the reactivity of the ions entering the collision/reaction cell with the reaction gas to be controlled. The obtained results showed that the He/H₂ mixture is not optimal for V determination in samples containing chlorides due to the insufficient suppression of the polyatomic interference of ³⁵Cl¹⁶O⁺. Data obtained from vanadium determination using the He/NH₃ mixture were consistent for all selected Cl⁻ concentrations, and the results were acceptable. The detection limit was comparable with detection limits obtained from ICP-MS equipped with a dynamic reaction cell. Analyses of elements forming interfering molecules, e.g., iron (⁵⁶Fe), arsenic (⁷⁵As) and selenium (⁸⁰Se), were in good agreement with the certified values for both studied collision/reaction gas mixtures.     

三维荧光光谱结合二阶校正方法测定细胞培养基中阿霉素的含量

本文采用交替三线性分解(ATLD)和交替归一加权残差三线性分解(ANWE)两种二阶校正方法结合激发发射矩阵荧光光谱对完全不经任何预处理的细胞培养基中的阿霉素进行简单、快速、直接的定量测定.当算法选取组分数为2时,解析得到细胞培养基中阿霉素的平均回收率分别为(100.5±1.8)%和(100.3±1.9)%.在细胞培养基中加入烟酰胺腺嘌呤二核苷酸(NADH)、烟酰胺腺嘌呤二核苷酸磷酸(NADPH)、黄素腺嘌呤二核苷酸(FAD)和黄素单核苷酸(FMN)四种细胞内的自发荧光物后,选取组分数为4时,解析得到细胞培养基中阿霉素的平均回收率分别为(99.1±2.9)%和(99.2±3.1)%.结果表明该分析方法能够准确、快速地直接测定细胞培养基中阿霉素的含量,并且在模拟细胞内荧光干扰环境下可定量测定阿霉素,且能获得令人满意的结果.     

From One‐Photon to Two‐Photon Probes: “Caged” Compounds,Actuators, and Photoswitches

Molecular systems that can be remotely controlled by light are gaining increasing importance in cell biology, physiology, and neurosciences because of the spatial and temporal precision that is achievable with laser microscopy. Two‐photon excitation has significant advantages deep in biological tissues, but raises problems in the design of “smart” probes compatible with cell physiology. This Review discusses the chemical challenges in generating suitable two‐photon probes.     

Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane

The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl‐PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl‐PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono‐dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology.     

Evaluation of matrix effect in one‐dimensional and comprehensive two‐dimensional liquid chromatography for the determination of the phenolic fraction in extra virgin olive oils

Olea europaea, meaning “European olive,” is a small tree belonging to the family Oleaceae, occurring in the Mediterranean Basin. Olive oil is an essential component of a balanced diet because of its nutritional value. Among micronutrients, phenolic compounds did show important beneficial effects for human health. The majority of the research studies on the phenol content are carried out by liquid chromatography combined to photodiode array and/or mass spectrometry detection; however, because of matrix complexity, one‐dimensional liquid chromatography cannot be sometimes sufficient to obtain rewarding separations, requiring more advanced analytical techniques. In this work, comprehensive two‐dimensional liquid chromatography, incorporating RP‐Amide and C18 stationary phases, in the first and second dimension, respectively, both under reversed phase conditions, was investigated for the determination of the phenolic fraction in extra virgin olive oil samples. As far as detection is concerned, triple quadrupole mass spectrometry was employed under multi reaction monitoring mode offering superior selectivity and sensitivity. The reduction of matrix effects, when using comprehensive two‐dimensional liquid chromatography with respect to conventional one‐dimensional liquid chromatography, was assessed by comparing the slopes of calibration curves built from standard solutions and spiked olive oil samples.     

Lysophosphatidic acid receptor 2 and Gi/Src pathway mediate cell motility through cyclooxygenase 2 expression in CAOV-3 ovarian cancer cells

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA-induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.     

Use of viral insecticides for pest control and production in cell culture

This review discusses a number of issues relevant to the production and use of baculoviruses for pest control. Following this review, patents pertaining to baculovirus use in pest control are summarized.