Influence of the microenvironment on gene and protein expression of odontogenic-like and osteogenic-like cells

Progenitor cells play an important biological role in tooth and bone formation, and previous analyses during bone and dentine induction have indicated that they may be a good alternative for tissue engineering. Thus, to clarify the influence of the microenvironment on protein and gene expression, MDPC23 cells (mouse dental papilla cell line) and KUSA/A1 cells (bone marrow stromal cell line) were used, both in vitro cell culture and in intra-abdominal diffusion chambers implanted in 4-week-old male immunodefficient mice (SCID mice). Our results indicate that KUSA/A1 cells differentiated into osteoblast-like cells and induced bone tissue inside the chamber, whereas, MDPC-23 showed odontoblast-like characteristics but with a low ability to induce dentin formation. This study shows that MDPC-23 cells are especial cells, which possess morphological and functional characteristics of odontoblast-like cells expressing dentin sialophosphoprotein in vivo. In contrast, dentin sialophosphoprotein gene and protein expression was not detected in both cell lines in vitro. The intra-abdominal diffusion chamber appears as an interesting experimental model for studying phenotypic expression of dental pulp cells in vivo.     

Regulation of gene expression for neurotransmitters during adaptation to hypoxia in oxygen-sensitive neuroendocrine cells

Reduced oxygen tension (hypoxia) in the environment stimulates oxygen-sensitive cells in the carotid body (CB). Upon exposure to hypoxia, the CB immediately triggers a reflexive physiological response, thereby increasing respiration. Adaptation to hypoxia involves changes in the expression of various CB genes, whose products are involved in the transduction and modulation of the hypoxic signal to the central nervous system (CNS). Genes encoding neurotransmitter-synthesizing enzymes and receptors are particularly important in this regard. The cellular response to hypoxia correlates closely with the release and biosynthesis of catecholamines. The gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is regulated by hypoxia in the CB and in the oxygen-sensitive cultured PC12 cell line. Recently, genomic microarray studies have identified additional genes regulated by hypoxia. Patterns of gene expression vary, depending on the type of applied hypoxia, e.g., intermittent vs. chronic. Construction of a hypoxia-regulated, CB-specific, subtractive cDNA library will enable us to further characterize regulation of gene expression in the CB.     

Calcium as a versatile second messenger in the control of gene expression.

The elevation of intracellular calcium is a major effector of stimulus-induced physiological change in a variety of cell types. Such change is invariably complex and frequently involves the activation of gene expression. Calcium signals are often able to activate different subsets of genes within the same cell, the basis for which has been unclear. Recent studies have revealed that a number of differing properties of the calcium signal are responsible for distinct cellular responses.     

In-Fusion克隆技术构建HBVX基因真核表达质粒

目的以血清中乙型肝炎病毒（HBV）DNA 为模板,克隆构建HBV X 蛋白质的真核表达载体pcDNA-HBx.方法：设计扩增HBV X 基因的In-Fusion PCR 引物,采用高保真DNA 聚合酶分两段扩增HBV X 基因序列;采用In-Fusion 克隆技术,将两段X 基因扩增片段-步克隆入经Hind Ⅲ 和Xba I 双酶切的pcDNA3.0 真核表达载体,以构建重组真核表达载体pcDNA-HBX ;采用Hind Ⅲ 和Xba I 双酶切和DNA 序列测序筛选鉴定重组载体;pcDNAHBx转染HepG2 细胞,Western blot 检测pcDNA-HBx 转染细胞的HBx 蛋白质表达.结果：In-Fusion PCR 引物分两段扩增获得全长HBx 基因序列;In-Fusion 获得克隆的pcDNA-HBx 经双酶切筛选得到阳性重组质粒,测序分析证实插入序列正确;转染pcDNA-HBX 的HepG2 细胞,Western blot 证实表达HBx 蛋白质.结论：以血清HBV DNA 为模板克隆HBV X 基因,构建了表达HBV HBx 蛋白质的真核表达载体,为HBx 蛋白质的生物学功能研究提供支持.     

Gap-junction quantification in biological tissues: freeze-fracture replicas versus thin sections

The relative efficiency of freeze-fracture replicas versus thin sections for the visualization and quantification of gap junctions in biological tissues has been evaluated. Both methods may underestimate gap-junction number—thin sections for reasons of tissue resolution and freeze-fracture replicas due to the mechanics of the fracturing process. Freeze-fracture misses gap junctions in regions of plasma membrane which are highly contoured, such as the overlapping basal cell processes of Drosophila imaginal wing discs and the interdigitating lateral membrane plications of intercalated discs in cardiac tissue. If the missed gap junctions are relatively large, as they are in both of these examples, freeze-fracture significantly underestimates the total gap-junctional area. Thin sections may miss small gap junctions, but in tissues which contain a range of gap-junction sizes the lost junctions constitute a relatively small fraction of the total junctional area. In neoplastic imaginal wing discs, thin sections were as efficient as freeze-fracture replicas in identifying even the smallest gap junctions. Although freeze-fracture may be the better technique for the qualitative and quantitative documentation of small gap junctions in tissues with relatively flat to gently contoured plasma membranes, and thin sections may be the superior method for gap-junction quantification in tissues containing a range of gap-junctional sizes and highly contoured cellular processes, the data suggest that a combination of the two approaches should be utilized whenever possible.     

In situ hybridization and immunofluorescence on resin‐embedded tissue to identify the components of Nissl substance

It is not clear whether the Nissl substance is present at the axon hillock. To clarify this gap in knowledge, we conducted in situ hybridization (ISH) on mouse brain tissue using 30‐μm cryostat and 1–3‐μm acrylic resin sections. Cryostat and rehydrated resin sections were exposed to digoxygenin‐labeled glutamic acid decarboxylase 1 sense and antisense riboprobes. Consecutive sections from tissue embedded in resin were subjected to the ribosomal protein L26 primary antibody to determine the distribution of the ribo/polysomes. ISH results from the antisense riboprobe in both cryostat and resin‐embedded tissue sections demonstrated an abundance of message in the neurons from the substantia nigra pars reticulate. In addition, the resin sections demonstrated hybridization signal in the axon hillock of some neurons. Immunofluorescence labeling of consecutive sections using an antibody to the most abundant ribosomal protein L26 confirmed their distribution in the cell body and the axon hillock of similar neurons. Compared with the 30‐μm cryostat sections, the most striking feature of ISH in the thinner resin (2–3 μm) sections was that there was a phenomenal improvement in the overall clarity and spatial resolution. Reexamination of the axon hillock when continuous with the cell body in cryostat sections revealed that the same message was also present, except it was overlooked initially because of overlapping cell populations in thick tissue slices. As ribosomes are a component of Nissl substance, we propose that the axon hillock, like other parts of the neuron, does contain Nissl substance. Microsc. Res. Tech. 2010. © 2009 Wiley‐Liss, Inc.     

Optimising adjacent membrane segmentation and parameterisation in multicellular aggregates by piecewise active contours

In fluorescence microscopy imaging, the segmentation of adjacent cell membranes within cell aggregates, multicellular samples, tissue, organs, or whole organisms remains a challenging task. The lipid bilayer is a very thin membrane when compared to the wavelength of photons in the visual spectra. Fluorescent molecules or proteins used for labelling membranes provide a limited signal intensity, and light scattering in combination with sample dynamics during in vivo imaging lead to poor or ambivalent signal patterns that hinder precise localisation of the membrane sheets. In the proximity of cells, membranes approach and distance each other. Here, the presence of membrane protrusions such as blebs; filopodia and lamellipodia; microvilli; or membrane vesicle trafficking, lead to a plurality of signal patterns, and the accurate localisation of two adjacent membranes becomes difficult. Several computational methods for membrane segmentation have been introduced. However, few of them specifically consider the accurate detection of adjacent membranes. In this article we present ALPACA (ALgorithm for Piecewise Adjacent Contour Adjustment), a novel method based on 2D piecewise parametric active contours that allows: (i) a definition of proximity for adjacent contours, (ii) a precise detection of adjacent, nonadjacent, and overlapping contour sections, (iii) the definition of a polyline for an optimised shared contour within adjacent sections and (iv) a solution for connecting adjacent and nonadjacent sections under the constraint of preserving the inherent cell morphology. We show that ALPACA leads to a precise quantification of adjacent and nonadjacent membrane zones in regular hexagons and live image sequences of cells of the parapineal organ during zebrafish embryo development. The algorithm detects and corrects adjacent, nonadjacent, and overlapping contour sections within a selected adjacency distance d, calculates shared contour sections for neighbouring cells with minimum alterations of the contour characteristics, and presents piecewise active contour solutions, preserving the contour shape and the overall cell morphology. ALPACA quantifies adjacent contours and can improve the meshing of 3D surfaces, the determination of forces, or tracking of contours in combination with previously published algorithms. We discuss pitfalls, strengths, and limits of our approach, and present a guideline to take the best decision for varying experimental conditions for in vivo microscopy.     

Effects of docosahexaenoic acid or arachidonic acid supplementation on gene expression and contractile force of rat cardiomyocytes in primary culture

While fatty acids play essential roles in the physiology of the myocardium, conventional culture media contain little lipid. We previously revealed that rat neonatal myocardium mainly contains docosahexaenoic (DHA), linoleic (LA), and arachidonic (AA) acids as polyunsaturated fatty acids (PUFAs), and these contents in cultured cardiomyocytes derived from fetal rats were markedly lower than those in the neonatal myocardium. In this study, we first assessed the effects of supplementation of DHA, LA, or AA on the fatty acid contents and the percentage change of contractile area in primarily cultured rat cardiomyocytes. Based on this assessment, we then evaluated the effects of DHA or AA supplementation on mRNA expression and further directly measured the contractile force of cardiomyocytes with the supplementations. This study revealed that percentage change of contractile area was maximized under 20 μM DHA or 50 μM AA supplementation while LA supplementation did not affect this contraction index, and that a widespread upregulation tendency of the mRNA expression related to differentiation, maturity, fatty acid metabolism, and cell adhesion was seen in the cultured cardiomyocytes with supplementation of DHA or AA. In particular, upregulation of the gene expression of cellular adhesion molecules connexin43 and N-cadherin were remarkable, whereas the effects on differentiation and maturation were less pronounced. Correspondingly, the increase of the percentage change of the contractile area of cardiomyocyte clusters in culture dishes with the supplementations was significant, whereas the enhancement of the contractile force was modest. These results suggest that supplementation of DHA or AA to the fetal cardiomyocyte culture may play effective roles in preventing the de-differentiation of the cardiomyocytes in culture and that the enhancement of the contractile performance may be mainly attributed to the improvement of intercellular connection.     

Toxicogenomic analysis of gene expression changes in human liver cells after exposure to environmentally friendly lubricants

A lubricant based on synthetic esters and a mixture of this lubricant with additives and metals simulating the intake caused by usage were investigated with various ecotoxicological and genotoxicological tests. Tests with algae, daphnids and bacteria demonstrated an influence of the mixture on ecotoxicity. The genotoxicity tests, however, showed no effects for both samples. In addition, genetic effects were examined in detail by using gene expression profiles of HepG2 cells. Comparison of the set of regulated genes for early and late time points indicated a certain concordance between the genes up‐regulated after 6 and 24 hours of exposure. The correlation for the corresponding down‐regulated gene groups is slightly lower. Generally, the number of regulated genes is low (3–6%) demonstrating a marginal influence. The results indicate that the assessment of gene expression profiles, in addition to the quantification of toxic effects, may give important information on ways of toxic action of a substance. These data can be used in the development of new chemicals or products in order to minimize toxic effects. Copyright © 2007 John Wiley & Sons, Ltd.     

Differential mRNA expression in peripheral blood is associated with oral squamous cell carcinoma: Recent advances and future challenges

Oral squamous cell carcinoma (OSCC) is a malignant tumor triggered by the accumulation of multiple gene mutations in oral epithelial cells. Different OSCC-related biomarkers have been reported in circulation in the peripheral blood that support the occurrence and development of OSCC. Recent advances in high-throughput and highly sensitive detection methods have overcome the limitation of the low concentration of most peripheral blood biomarkers. Hence, blood biomarker detection has become an efficient screening tool for the early diagnosis of OSCC. The growing data available in public cancer and gene databases have provided new foundations for OSCC research. In particular, the identification of OSCC biomarkers using bioinformatic tools has shed new light on the underlying mechanisms as well as on the genetic landscape of OSCC. More recently, mRNA targeting therapies have emerged as valuable anticancer treatment strategies, as they allow for the regulation of the expression of certain functional proteins to reverse genetic abnormalities or induce tissue repair. Thus, mRNA-targeting therapies can be used to regulate the expression of antigens, antibodies, or cellular receptors by immune cells. Particularly, anti-cancer cellular immunotherapy carrying specific mRNAs has attracted significant attention in OSCC treatment. Here, we review the present knowledge on the role of peripheral blood mRNAs in the diagnosis, treatment, development, and prognosis of OSCC. Moreover, we address future research prospects of mRNAs in the peripheral blood in OSCC and the opportunities and challenges that may arise in future clinical therapeutic applications.     

实时定量PCR分析PtDrl01基因hpRNA干扰下的表达

PtDrl01基因从三倍体毛白杨[（Populus tomentosa×P.bolleana）×P.tomentosa]中克隆获得，能够编码NBS-LRR型蛋白，受到水杨酸和甲基茉莉酸诱导表达，是一种广谱的抗病基因。本研究利用实时定量PCR技术分析转PtDrl01基因hpRNA干扰载体的表达模式，分析发现4个表达模式显著差异的转基因株系，为进一步功能鉴定提供材料。本研究表明实时定量PCR能够高灵敏度、精确地检测hpRNA干扰情况下的基因表达量。     

Reflectance in situ hybridization (RISH): detection,by confocal reflectance laser microscopy,of gold-labelled riboprobes in breast cancer cell lines and histological specimens

A method for reflectance in situ hybridization (RISH) is presented. The importance of the method is demonstrated by results obtained on cytological and histological breast cancer specimens. Scattering reflectance signals from 1-nm colloidal-gold particles after RNA/RNA in situ hybridization, using digoxigenin-labelled riboprobes, were detected by confocal scanning laser microscopy. The mRNA expression of two ras-related genes, rho B and rho C, was analysed in human histological breast cancer specimens and in human breast cancer cell lines. Horizontal (x, y) and vertical (z) optical sections after three-dimensional imaging were used for visualization. A marked heterogeneity (between individual cells and between specimens) was noted for the expression of the rho B gene, both in cytological and in histological samples. On the other hand, rho C was always expressed and showed no heterogeneity. This method allows the identification of several cellular constituents in an heterogeneous tissue structure, as demonstrated by the simultaneous detection of rho B (or rho C) by reflectance and of DNA, cytokeratin and/or vimentin by fluorescence.     

Morphology and expression status investigations of specific surface markers on B‐cell chronic lymphocytic leukemia cells

The morphology of cells and expression status of specific surface markers [cluster of differentiation (CD)], such as CD5, CD19, CD20, CD38, and CD45, have long been considered as the essential indicators for the diagnosis and prognosis of B‐cell chronic lymphocytic leukemia (B‐CLL). Clinically, it is difficult to simultaneously obtain cell morphology and distribution of surface markers with flow cytometry, especially for some surrogate markers such as CD38. Here, as an alternative and complementary prognostic method, fluorescence microscopy and image processing method are introduced to directly visualize the cells from patients and to quantitatively determine the expression status of surface markers. In this study, the morphological parameters of B‐CLL cells were measured to establish the correlation between the cellular morphology and the surface marker expression. It was clear that the CD38+ and CD38? B‐CLL cells from the same CD38+ patients had hardly any size differences; however, an increase in perimeter was observed for CD38? patients. Moreover, the expression level of the receptors on the cell was independent of the cell size. There was no evidence showing that the expression intensities of CD19 and CD38 were related to each other for the CD38+ B‐CLL cells. On the same cells, CD5 was more selectively expressed on the cell membrane; however, the expression patterns suggested that the cell membrane of CD38? B‐CLL cells contained the least expression level of CD19. Microsc. Res. Tech. 76:1147–1153, 2013. © 2013 Wiley Periodicals, Inc.     

Quantification of substrate and cellular strains in stretchable 3D cell cultures: an experimental and computational framework

The mechanical cell environment is a key regulator of biological processes . In living tissues, cells are embedded into the 3D extracellular matrix and permanently exposed to mechanical forces. Quantification of the cellular strain state in a 3D matrix is therefore the first step towards understanding how physical cues determine single cell and multicellular behaviour. The majority of cell assays are, however, based on 2D cell cultures that lack many essential features of the in vivo cellular environment. Furthermore, nondestructive measurement of substrate and cellular mechanics requires appropriate computational tools for microscopic image analysis and interpretation. Here, we present an experimental and computational framework for generation and quantification of the cellular strain state in 3D cell cultures using a combination of 3D substrate stretcher, multichannel microscopic imaging and computational image analysis. The 3D substrate stretcher enables deformation of living cells embedded in bead‐labelled 3D collagen hydrogels. Local substrate and cell deformations are determined by tracking displacement of fluorescent beads with subsequent finite element interpolation of cell strains over a tetrahedral tessellation. In this feasibility study, we debate diverse aspects of deformable 3D culture construction, quantification and evaluation, and present an example of its application for quantitative analysis of a cellular model system based on primary mouse hepatocytes undergoing transforming growth factor (TGF‐β) induced epithelial‐to‐mesenchymal transition.     

Gene expression profile analysis reveals the effect of metformin treatment on HepG2 cells

Metformin is a first-line drug in the fight against type 2 diabetes. In recent years, studies have shown that metformin has some preventive and therapeutic effects on liver cancer, but the effects of metformin on the gene expression of liver cancer cells are not fully known. This study focused on the differences in the gene expression profiles in liver cancer cells treated with or without metformin. A total of 153 differentially expressed genes (DEGs) (FC > 2 and q-values < 0.001) were found, including 77 upregulated genes and 76 downregulated genes. These DEGs are involved in mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), cell adhesion molecules (CAMs), and leukocyte transendothelial migration signaling pathways. These findings reveal the effects of metformin treatment on gene expression profiles in liver cancer cells and provide new clues for unveiling the mechanism of the antitumor effects of metformin.     

A semiautomated image analysis procedure for the quantification of dental microwear

Different diets have been shown to leave characteristic patterns of microwear on the teeth of various mammalian species. The quantification of microwear features facilitates the comparison of taxa, and allows the discrimination of wear patterns associated with specific diets. The current study reports on a semiautomated image analysis procedure designed to expedite microwear quantification by engaging an IBM PS/2 running OS/2 Presentation Manager to compute and record the density, dimensions, and orientations of features defined by a user employing a mouse-driven cursor. This technique is used to distinguish microwear patterns on the maxillary central incisors (I¹s) of four Sumatran primate species (n=5 per taxon) with known dietary differences (Hylobates lar, Macaca fascicularis, Pongo pygmaeus, and Presbytis thomasi). However, although developed for a particular problem area, the analytical routines described are of general interest.     

Muscle-specific gene expression during myogensis in the mouse

Over the past decade, significant advances in molecular biological techniques have substantially increased our understanding of in vivo myogenesis, supplementing the information that previously had been obtained from classical embryological and morphological studies of muscle development. In this review, we have attempted to correlate morphogenetic events in developing murine muscle with the expression of genes encoding the MyoD family of myogenic regulatory factors and the contractile proteins. Differences in the pattern of expression of these genes in murine myotomal and limb muscle are discussed in the context of muscle cell lineage and environmetal factors. The differences in gene expression in these two types of muscle suggest that no single coordinated pattern of gene activation is required during the initial formation of the muscles of the mouse. © 1995 Wiley-Liss, Inc.