Quantitative microspectrometrical determination of the total protein and total protein-SH content of the nuclei of Ehrlich ascites tumor cells without prior isolation

The total protein and total protein-SH content of both the cytoplasmic fraction and the nuclei of Ehrlich ascites tumor cells (EATC) were determined macroscopically and microspectrometrically. The separation into cytoplasmic and nuclear fraction was performed by a modification of the method of Mamaril et al. [4]. The macroscopical determination of the total protein and the total protein-SH content were performed with the Folin method of Lowry et al. [2] and the DTNB method of Ellman [1], respectively. The quantitative microspectrometrical determinations of total protein content and of total protein-SH content were performed using the tetrazonium staining method of N?hammer [5] and N?hammer et al. [6] and the mercurochromcyanide (MCN) method of N?hammer et al. [7], respectively. Within the intact cells, fixed and stained histochemically, the total protein and the total protein-SH content of the nuclei were determined microspectrometrically. The so called "nuclear extinctions", measured as the sum of the extinctions of the nucleus and of the parts of the cytoplasm above and below the nucleus, were calculated into the true nuclear extinctions, which then show a good correspondence to the values measured both microspectrometrically and macroscopically on isolated nuclei. The calculation for the true nuclear extinctions is based on a special preparation of spherically shaped cells and nuclei.     

Characterization of squamous cell carcinomas of the head and neck using methods of spatial statistics

In the present study, 53 cases of squamous cell carcinomas of the head and neck were characterized by a quantitative histological texture analysis based on principles of spatial statistics. A planar tessellation of the epithelial tumour component was generated by a skeletonization algorithm. The size distribution of the virtual cells of this planar tessellation, and the size distribution of the profiles of the tumour cell nuclei were estimated in terms of area and boundary length. The intensity, the reduced second moment function (K‐function) and the pair correlation function of the point process of the centroids of the profiles of the tumour cell nuclei were also estimated. For both purposes, it is necessary to correct for edge effects, which we consider in this paper in some detail. Specifically, the point patterns of the tumour cell nuclei were considered as realizations of a point process, where the points exist only in the epithelial tumour component (the permitted phase) and not in the stroma (the forbidden phase). The methods allow to characterize each individual tumour by a series of summary statistics. The total set of cases was then partitioned into two groups: 19 cases without lymph node metastases (pN0), and 34 nodal positive cases (pN1 or pN2). Statistical analysis showed no significant differences between the intensities, the mean K‐functions and the mean pair correlation functions of the tumour cell nucleus profiles of the two groups. However, there were some significant differences between the sizes of the virtual cells and of the nucleus profiles of the nodal negative cases as compared to the nodal positive cases. In a logistic regression analysis, one of the quantitative nuclear size variables (mean nuclear area) was found to be a significant predictor of lymph node metastasis, in addition to tumour stage. The study shows the potential of methods of spatial statistics for objective quantitative grading of squamous cell carcinomas of the head and neck, and provides an example for modelling histological point patterns as realizations of planar point processes occupying a reference phase which is only a partial component of the total tissue.     

Iron oxide nanoparticles decrease nuclear fractal dimension of buccal epithelial cells in a time‐dependent manner

In this paper, we present results that iron oxide nanoparticles (IONPs) induce time‐dependent structural changes in nuclei of buccal epithelial cells. The cells were treated with magnetite, Fe₃O₄ nanoparticles (spherical shape, diameter 80–100 nanometres). The digital micrographs of the nuclei were made in 3 different time points: 15, 30 and 60 min after the treatment with IONPs, as well as in the control cells. A total of 120 nuclear structures (30 per sample) were analysed. Fractal analysis of nuclei was done in ImageJ software of the National Institutes of Health, (Bethesda, MD, USA). For each nuclear structure, the values of fractal dimension and lacunarity were calculated. There was a time‐dependent reduction of nuclear fractal dimension in buccal epithelial cells after exposure to magnetite iron oxide nanoparticles. Negative trend was observed (p < 0.01). Nuclear lacunarity, as another fractal parameter was shown to increase, also in a time‐dependent manner, after the treatment with IONPs. To our knowledge, this is the first study to investigate effects of magnetite nanomaterial on nuclear fractal complexity, and also the first to apply fractal analysis method in testing of the interaction between nanoparticles and cell nucleus in this experimental setting.     

中碳多元合金钢破碎机锤头的研制

对采用Cr、Mo、Ni及Mn等元素多元合金化中碳合金钢锤式破碎机锤头的组织、力学性能及耐冲击磨损性能进行了试验研究。锤头采用自硬树脂砂工艺铸造 ,对所研制的锤头进行了现场装机试验。试验结果表明 :合金元素含量分别为 :w(Cr) =4～ 5 %、w(Mo) =0 5～ 0 7%、w(Ni) =0 3～ 0 5 %、w(Mn) =0 6～ 0 9% ,含碳量为 :w(C) =0 4～ 0 5 %的中碳多元合金钢锤头具有高耐冲击磨损和良好韧性相结合的特点 ,其工作寿命为高锰钢锤头的 1 5 5倍     

KIF18A is a potential prognostic factor and promotes tumor progression in oral tongue squamous cell carcinoma

The kinesin family member 18A protein was dysregulated in several human cancers and involved in cancer progression. However, the significance in oral tongue squamous cell carcinoma (OTSCC) has not been studied. The present study was intended to explore the functions of KIF18A in oral tongue squamous cell carcinoma. The immunohistochemistry (IHC) assay was performed to assess the relationships between the KIF18A protein expression level and clinical-pathological features of the patients. The biological functions of KIF18A in OTSCC cells were investigated by the experiments in vitro and in vivo. Based on immunohistochemistry, we found that KIF18A was correlated with the clinical-pathological features of OTSCC patients. High expression of KIF18A was associated with the lymph node metastasis, and clinical stages. In vitro experiments revealed that silencing of KIF18A significantly inhibited the expression of the proliferation and migration related proteins such as Ki67, proliferating cell nuclear antigen, matrix metalloproteinase-7 and matrix metalloproteinase-9, and thereby inhibiting the proliferation, migration and invasion of tumor cells. In vivo, knocking-down of KIF18A could inhibit the tumor growth in nude mice. In conclusion, we found KIF18A promoted tumor progression in vivo and in vitro and might become an effective target for the treatment of OTSCC.     

HMGB1 promotes the proliferation and invasion of oral squamous cell carcinoma via activating epithelial-mesenchymal transformation

This study aimed to investigate the role of high mobility group box-1 (HMGB1) expression in oral squamous cell carcinoma; HMGB1 promoted the proliferation and invasion of oral squamous cell carcinoma via activating epithelial-mesenchymal transformation (EMT). In this study, RNA transfection was used to silence the expression of HMGB1 in oral squamous cell carcinoma cells. CCK-8, cell clone formation and trans-well assays were used to detect the proliferation and invasion of cells before and after HMGB1 silencing. qRT-PCR and Western blot were used to detect changes in EMT marker protein expression before and after transfection. HMGB1 was significantly higher in OSCC tissues than in adjacent tissues, and of the cell lines examined, HMGB1 was highest in SCC-9 cells. Additionally, HMGB1 silencing decreased SCC-9 cell proliferation and viability. Down-regulation of HMBG1 expression inhibited not only the proliferation but also the invasion of SCC-9 cells. The expression of N-cadherin, Snail, and Slug, but not E-cadherin, were promoted after silencing HMGB1. The expression of HMGB1 in OSCC tissue and cell lines was higher, and HMGB1 silencing decreased SCC-9 cell proliferation and invasion, suggesting that HMGB1 has positive effects on OSCC development. Down-regulation of HMBG1 expression regulates EMT markers, suggesting that HMBG1 promotes OSCC cell proliferation and invasion is likely to be associated with EMT activation.     

Estimation of the mean caliper diameter of cell nuclei. I. Serial section reconstriction method and endothelial nuclei from human lung.

In morphometric studies of lung tissue, accurate determination of the total number of any specific type of cell, such as the endothelial cell, requires knowledge of the shape of the cell nucleus. This knowledge of shape is necessary to calculate the mean caliper diameter, which, in turn, is an indispensible element of the equation for the number of nuclei (therefore cells) per unit volume (NV). Nine human hung endothelial cell nuclei were therfore reconstructed in dental wax from serial sections and were found to be pleomorphic triaxial ellipsoids. Five of these approached an oblate shape. The axes of these nine nuclear models were directly measured and a computer program using numerical integration was written to determine the mean caliper diameters of these ellipsoids. The estimated mean semi-axes of the nine nuclei were (+/-SD) 5.98+/-1.61, 3.61+/-0.70, 1.36+/-0.34; and the estimated overall mean caliper diameter for the total population of human lung endothelial nuclei was 7.97+/-1.27 micrometers.     

Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.     

Confocal microscopy as a tool for the study of the intranuclear topography of chromosomes

A scanning confocal microscope was used to investigate the spatial positions of specific regions within blood cell nuclei. These centromeric regions were fluorescently labelled by in-situ hybridization to suspended nuclei with a centromere-1-specific DNA probe. The 3-D image data sets, obtained by optical sectioning of the cells, were used to determine the spatial position of the centromeric regions in the nuclei by means of specially developed software. The centromeres were found to be localized near the nuclear boundary. This spatial pattern was tested against a random distribution model by means of the Kolmogorov—Smirnov test. The difference between the two patterns was at a P < 0?01 significance level.     

Use of an automated image analyser to quantitate cellular hyperplasia in urinary bladder epithelium.

The feasibility of using an automated image analyser to evaluate quantitatively hyperplasia caused by N-butyl-N-(4-hydroxybutyl) nitrosamine in bladder epithelium was studied. The number of cells per unit length of epithelium was counted manually, and compared with automated measurements of: (1) the number of nuclei, (2) the number of positive tangents to the lower edge of nuclei, (3) the nuclear cross-sectional area, and (4) the epithelial cross-sectional area per unit length respectively. Regression of each of the automated measurements on the manual counts all revealed close linear relationships with correlation coefficients in excess of 0-9. Coefficients of variation for repetitive automated measurements were less than or equal to 0-06 in each of the four modes. The automated system resulted in a great saving in time over manual counting. It is concluded that the automated image analyser provides an accurate, precise, and efficient tool for estimating epithelial cell numbers in normal and hyperplastic bladder epithelia.     

Immunogold labelling of the intermediate filament-lamina-nuclear matrix system in hela and bhk-21 cells

Whole-mount, sequentially extracted cells combined with immunogold electron microscopy were developed to demonstrate the intermediate filaments, lamina, and nuclear matrix (IF-L-NM) and to identify their protein components. The IFs of HeLa cells were reacted both with keratin and vimentin monoclonal antibodies; meanwhile, the IF network of BHK-21 cell was reacted only with vimentin monoclonal antibody. The lamina and nuclear matrices of both HeLa and BHK-21 cell were labelled, respectively, with lamin monoclonal antibody-gold complex and 280 Kd nuclear matrix protein monoclonal antibody-gold complex. The monoclonal antibody to keratin could cross-react with the lamina both of HeLa and BHK-21 cells.     

Determination of the mean caliper diameter of lung nuclei by a method which is independent of shape assumptions

When determining the number of nuclei (therefore cells) present in a known volume of tissue it is necessary to determine the overall mean caliper diameter of nuclei for each cell type studied. Most previous methods for determining the mean caliper diameters (D?) of cell nuclei were strongly dependent upon assumptions of the shape of the particles in question (Greeley et al., 1978). A computer program has now been designed to calculate D? from three-dimensional reconstructions of nuclei in a manner that is independent of all shape assumptions. The D?s of ten capillary endothelial cell nuclei from normal rat lungs have been determined by this shape-independent method. The resulting overall mean caliper diameter for this group of ten nuclei varied only slightly from that previously estimated for them by a method which assumes them to be triaxial ellipsoids. In a similar comparison, the D?s for ten normal human lung endothelial cell nuclei were determined by both methods. The overall mean caliper diameter determined by the shape-independent method was 10% larger than that estimated by the shape-dependent method. This suggests that human lung endothelial nuclei vary significantly from the shape assumption (triaxial ellipsoid) employed in the shape-dependent method, and the shape-independent method was considered to yield a more accurate measurement of D?.     

Counting cells with stereology: Random versus serial sectioning

Counts of cells and nuclei from sections provide information central to studying structural changes in cells, tissues, and organs. This study considers some of the practical problems associated with counting cells with the newer random and serial sectioning methods of stereology and tests the hypothesis that similar cell counts can be obtained with both random and serial sectioning methods. Using irregularly shaped nuclei from alveolar cells of the goat lung, we compared cell counts derived from random (electron microscopic) and serial sectioning (light microscopic) methods. The results showed that both sectioning methods gave similar cell counts (10⁷/cm³ of parenchyma) for type 1 epithelial cells (5.0 vs. 5.0; P=1.0), type 2 epithelial cells (8.6 vs. 9.8; P=0.42) and interstitial cells (34.6 vs. 33.4; P=0.64), provided that corrections were introduced for sectionrelated biases and that the nuclei of the random sectioning method were corrected for shape. We found counting biases of 5%–7% for nuclear shape and 16% for section compression. These observations support the hypothesis that similar cell counts can be obtained with random and serial sectioning, even when nuclei have irregular shapes.     

体外培养的不同亚型肺癌细胞株差异蛋白的初步分析

分析体外培养的不同亚型肺癌细胞株蛋白质表达差异,筛选肺癌细胞的标志蛋白并与肺癌病人血清中的标志蛋白进行对比分析。采用SELDI(Surface Enhanced LaserDesorption/Ionization)蛋白质芯片技术检测了三种肺癌细胞株A549(肺癌)、Calu-6(腺癌)和PG(大细胞癌)以及人胚肺二倍体成纤维细胞(2BS)的蛋白质谱。结果显示与2BS细胞比较,肺癌细胞有24个蛋白质表达发生明显改变。     

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.     

Characterization of Sertoli cell perinuclear filaments.

Sertoli cell nuclei are characterized by deep invaginations and, in addition, the orientation of the nuclei with respect to the wall of the seminiferous tubules varies during the cycle of the seminiferous epithelium. These events may be the result of cytoplasmic filaments acting at the level of the nuclear capsule and may represent significant changes in Sertoli cell activity. Thus, a study was performed to characterize the nature of the perinuclear filaments of Sertoli cells in vivo and in vitro. In Sertoli cells in vivo, microtubules and microfilaments were often detected in the perinuclear cytoplasm, and these cytoskeletal components were observed to course either parallel to, or abut at, the nuclear capsule. In Sertoli cells in vitro, the nuclear infoldings are retained and the perinuclear cytoskeleton was shown to contain microtubules, f-actin, and intermediate filaments. A fixation-permeabilization protocol employing tannic acid-saponin was used and it significantly enhanced the preservation of cytoskeletal components. The presence of f-actin was demonstrated by using the S1 fragment of muscle myosin to decorate the microfilaments. Treatment of the cultured cells with either microtubule or f-actin depolymerizing agents had no effect on nuclear shape. Thus, at present, the function of the prominent perinuclear cytoskeletal components remains unknown.     

Immuno‐detection of mRNA‐binding protein complex in human cells under transmission electron microscopy

Transit from the nuclear complex to the cytoplasm through the nuclear pore complex permits modification of mRNA, including processing such as splicing, capping, and polyadenylation, etc. At each of these events, mRNA interacts with various proteins to form mRNA‐protein complex. Visualizing the mRNA is crucial for understanding the mechanisms underlying mRNA processing and elucidating its structure and recent advances in mRNA imaging allow detection of real‐time mRNA localization in living cells. However, these techniques revealed only the location of mRNA but cannot visualize the conformation of mRNA‐protein complex in cells. On the other hand, transmission electron microscopy has been used to visualize the structure of the Balbiani ring‐derived large mRNA, but their observations were limited to the insect cells. In this study, we visualized the structure of mRNA‐protein complex in human culture cells by using immuno‐electron microscopy. Through immuno‐detection, an mRNA exon junction binding complex Y14, and its binding protien Upf2, different gold particle patterns were imaged with transmission electron microscopy and analyzed. Characteristic linear and stacked particle orientation were observed. Across the nuclear membrane, only linear aggregation pattern was observed, whereas the stacked aggregation pattern was detected in the cytoplasm. Our method is able to visualize mRNA‐conformation and applicable to many cell types, including mammalian cells, where genes can easily be manipulated.     

The development of squamous cell metaplasia in human bronchial epithelium by light microscopic morphometry

Bronchial biopsies from ten subjects, including five smokers, have been examined using light microscopic morphometry. The biopsies were free from identifiable disease. Using manual point counting and a Quantimet 720 image analysing computer, a number of parameters were measured. Computer-based cluster analysis of seven of these parameters associated the subjects into three groups: visual inspection of the sections achieved the same separation. Four subjects (non-smokers) had normal epithelia: four subjects (one non-smoker, three smokers) showed mucous cell hyperplasia: two subjects (smokers) had squamous cell metaplasia. Three parameters in conjunction contained sufficient information to characterize accurately the histological appearance of the epithelia: the epithelial thickness, the volume density of intracellular mucus and the number of nuclear profiles per unit area of sectioned epithelium. Reduction of these three parameters to a linear plot closely approximated a similar reduction of the original seven parameters. These three parameters can be measured rapidly, either manually or by the Quantimet. The linear representation of these parameters provides a reproducible and objective basis for comparing specimens of bronchial epithelium.     

Plakophilin, armadillo repeats, and nuclear localization

Plakophilins are armadillo-repeat containing proteins, identified through their localization to desmosomes. Expressed in a wide range of tissues, plakophilins are largely nuclear in most cell types [Schmidt et al. (1997) Cell Tissue Res 290:481; Mertens et al. (1996) J. Cell Biol 135:1009]. Using Xenopus embryos and cultured A6 cells, together with myc- and green fluorescent protein (GFP)-tags, we found that both the N-terminal, non-armadillo repeat "head" and the C-terminal armadillo repeat-containing regions can enter nuclei. The "arm" repeat domain is predominantly cytoplasmic and concentrated at the cell cortex, whereas the head and full-length polypeptides are concentrated in the nucleus. The head domain can also be seen to decorate and disrupt keratin filament network organization in some cells. In the course of these studies, we found that the distribution of the myc-epitope and green fluorescence differed in fixed cells, e.g., while the green fluorescence of a myc- and GFP-tagged head domain polypeptide was usually exclusively nuclear, a substantial fraction of the myc-immunoreactivity was cytoplasmic. Treating cells with the translation inhibitor cycloheximide reduces the cytoplasmic myc-signal, suggesting that it represented nascent polypeptides awaiting folding and nuclear import. Based on these types of experiments, GFP can be seen as a marker of the distribution of the mature form of the tagged polypeptide.