Comparison of smears and cell blocks in the fine needle aspiration diagnosis of recurrent gynecologic malignancies.

A retrospective, seven-year study was conducted to evaluate the value of cell blocks as an adjunct to smears in the fine needle aspiration (FNA) diagnosis of recurrent gynecologic malignancies. Eighty-four FNAs were performed on patients with previously diagnosed malignancies of the cervix (39 cases), ovary (27), uterus (14), vulva (2) and vagina (2). Material for the preparation of cell blocks was available in all cases. Smears and cell blocks were reviewed separately, and the findings were categorized as positive, negative, suspicious or unsatisfactory. Identical smear and cell block results were reported in 71 (84.5%) of the 84 cases (45 positive, 20 negative, 1 suspicious and 5 unsatisfactory). In 12 cases (14.3%) the smear was superior to the cell block in detecting malignant cells; while all 12 smears were positive, 8 cell blocks were negative, and 4 were suspicious. In no case was the cell block positive with a negative smear; in one (1.2%) the cell block was positive and the smear suspicious. The results of this study indicate that the additional study of cell blocks is of little benefit in the FNA cytodiagnosis of recurrent disease in patients with documented gynecologic malignancies.     

Electrical perception of "death message" in chara: analysis of rapid component and ionic process

Electrical response upon wounding was analyzed in Chara corallina. A specimen comprising two adjoining internodal cells was prepared. One cell (victim cell) was killed by cutting and any changes in the membrane potential of the neighboring cell (the receptor cell) were measured. Upon cutting the victim cell, the receptor cell generated four kinds of depolarizations: (1) rapid component, (2) slow and long-lasting component, (3) action potential and (4) small spike. Rapid and slow components were observed in most cells. On the other hand, the action potential and small spike were not always ubiquitous among specimens. When an action potential was generated just after cutting the victim cell, the rapid component could not be observed due to masking by the action potential. It was suggested that both rapid and slow components were generated at the nodal end. On the other hand, action potentials were thought to be generated at the flank of the receptor cell. High turgor pressure of the cell was necessary for generating both rapid and slow components. Experiments under K(+)-induced depolarization unequivocally showed that the Cl(-) channel at the nodal end of the receptor cell was activated upon cutting the victim cell.     

Cell-to-cell communication in response of E. coli cells at different phases of growth to low-intensity microwaves

Effects of millimeter waves (MMW) at the frequency of 51.755 GHz were studied in logarithmic and stationary E. coli cells at various cell densities. The changes in the genome conformational state (GCS) were analyzed by the method of anomalous viscosity time dependence (AVTD). Before lysis, the cells were adjusted to the cell density of 4x10(7) cells/ml and all AVTD measurements were run at this cell density. Stationary cells responded to MMW by increase in AVTD, while the same MMW exposure decreased AVTD in logarithmic cells. MMW effects depended on cell density during exposure and were stronger for stationary cells. The observed dependence on cell density suggested a cell-to-cell communication between cells during exposure to microwaves. Decrease in power density (PD) resulted in more striking differences between responses at different cell densities. The data provided evidence that intercellular communication in response to MMW depended on cell status and PD of microwaves. The MMW effects were studied in more detail at low intensity of 10(-17) W/cm(2) in the range of cell densities 4x10(7) to 8x10(8) cells/ml. The obtained sigmoid-like dependence of MMW effect on cell density saturated at approximately 5x10(8) cells/ml. The dependence of MMW effect on cell density was very similar in this study and in previous studies with weak extremely low frequency (ELF) electromagnetic fields (EMF). The data suggested that cell-to-cell communication might be involved in response of cells to weak EMF of various frequency ranges.     

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.     

Evaluating the biological effects of intermittent 1.9 GHz pulse-modulated radiofrequency fields in a series of human-derived cell lines

Several recent studies have suggested that radiofrequency (RF) fields may cause changes in a variety of cellular functions that may eventually lead to potential long-term health effects. In the present study, we have assessed the ability of non-thermal RF-field exposure to affect a variety of biological processes (including apoptosis, cell cycle progression, viability and cytokine production) in a series of human-derived cell lines (TK6, HL60 and Mono-Mac-6). Exponentially growing cells were exposed to intermittent (5 min on, 10 min off) 1.9 GHz pulse-modulated RF fields for 6 h at mean specific absorption rates (SARs) of 0, 1 and 10 W/kg. Concurrent negative (incubator) and positive (heat shock for 1 h at 43 degrees C) controls were included in each experiment. Immediately after the 6-h exposure period and 18 h after exposure, cell pellets were collected and analyzed for cell viability, the incidence of apoptosis, and alterations in cell cycle kinetics. The cell culture supernatants were assessed for the presence of a series of human inflammatory cytokines (TNFA, IL1B, IL6, IL8, IL10, IL12) using a cytometric bead array assay. No detectable changes in cell viability, cell cycle kinetics, incidence of apoptosis, or cytokine expression were observed in any of RF-field-exposed groups in any of the cell lines tested, relative to the sham controls. However, the positive (heat-shock) control samples displayed a significant decrease in cell viability, increase in apoptosis, and alteration in cell cycle kinetics (G(2)/M block). Overall, we found no evidence that non-thermal RF-field exposure could elicit any detectable biological effect in three human-derived cell lines.     

New approach for the establishment of mouse early embryonic stem cells and induction of their differentiation

Eleven early embryonic stem (EES) cell lines were established using a new novel method. Two cell stage embryos from the ddY mouse strain were cultured in alpha-MEM supplemented with 10% fetal calf serum (FCS) and embryotrophic factors (ETFs) and allowed to develop to the trilaminal germ disc embryonic stage. Only small round cells (EES cells) were isolated by the colony isolating technique and subsequently cultured in the same medium containing the ETFs and leukemia inhibitory factors (LIF-10 ng/ml). The newly established embryonic stem (ES) cells isolated from inner cell mass of blastocysts differentiated from two cell stage embryo in culture. The EES and ES cell lines were maintained in an undifferentiated state using Ham's F12 medium supplemented with 10% FCS and 1 ng/ml of LIF. The EES cells maintained their normal genetic and morphological features as well as their potential to differentiate into a broad spectrum of cell types as well as their ability to contribute to all cell lineages in chimeric mice. Moreover, these cell lines changed and differentiated into various kinds of cells by removing LIF and by the addition of ETFs to the vitro culture system. All 11 EES cell lines and 3 ES cell lines formed embryoid bodies; however, cell line EES-4 formed tube-like structures which extended, anastomosed with each other, and finally formed networks when the LIF were absent. Primitive germ organ-like structures composed of 3 germ layers were recognized in the cultures following the administration of ETFs. In conclusion, the new method devised by us is a novel, easy and reliable technique for establishing EES cell lines.     

抗汉坦病毒核蛋白细胞内抗体抗病毒作用的研究

本文报道在内质网和细胞浆内稳定表达抗汉坦病毒核蛋白细胞内抗体,研究抗汉坦病毒核蛋白细胞内抗体抗病毒作用及其抗病毒作用机理。在获得稳定表达抗汉坦病毒核蛋白细胞内抗体的细胞系的基础上,用MTT法检测细胞内表达抗汉坦病毒单链抗体对细胞增殖的影响,证实细胞内抗体的表达不会影响到细胞的生长。不同时间收集病毒感染毒后细胞培养培养上清和细胞裂解物,用ELISA方法检测细胞内外病毒结构蛋白含量的变化,结果表明定位于细胞内质肉和细胞浆内的抗汉坦病毒核蛋白细胞内抗体均能不同程度降低感染细胞上清中的核蛋白含量,但是不能或轻微抑制细胞内汉坦病毒核蛋白的合成。利用病毒微量滴定免疫荧光方法,对细胞内抗体与病毒的增殖关系进行了研究,证实无论在内质网还是在胞质,抗核蛋白细胞内抗体都能够抑制病毒的增殖,具有抗病毒活性。抗汉坦病毒核蛋白细胞内抗体不能抑制病毒结构蛋白的合成,其抗病毒效果是通过抑制病毒的包装而实现。     

Immunospecific labeling of mouse lymphocytes in the scanning electron microscope

Bone marrow-derived (B) and thymus-derived (T) Balb/c mouse lymphocytes were identified in the scanning electron microscope (SEM) by the immunospecific attachment of one of several kinds of large-molecular-weight markers distinguishable in SEM. These markers (tobacco mosaic virus, keyhole limpet hemocyanin, bushy stunt virus, and bacteriophage T4) could be modified with hapten groups and linked with anti-hapten antibody, in an indirect (sandwich) scheme, to hapten-modified anti-cell-surface antibody bound to the cell surface. Hapten-modified antibodies to B cell antigens (goat anti-mouse-immunoglobulin) or to T cell antigens (rabbit anti-mouse brain) were employed to identify these two lymphoid cell types in unfractionated spleen, mesenteric lymph node, bone marrow, and thymus cell populations. The topography of B cells was always indistinguishable from that of T cells. No surface features were found to be unique to either cell type. In suspension, the majority of B and T cells had one or no microvilli regardless of the tissue source of the labeled cells. Cells in suspension that had microvilli (usually 10% of the total cell population) were always unlabeled. However, after cell contact with a glass surface, approximately half of both the B and T cell populations had a villous topography.     

Development and testing of a bovine trichomoniasis vaccine

Eighteen culled dairy cows were randomly allocated into 1 of 5 treatment groups. Six cows were vaccinated twice (2V), 21 days apart, 3 with whole cell (2WC) and 3 with fragmented cell membrane (2FC) containing 1 x 10(9)Trichomonas fetus organisms or protein equivalent in a commercial mineral oil adjuvant vaccine. Six more cows were vaccinated once (1V), 3 with whole cell (1WC) and 3 with fragmented cell vaccine (1FC), using the same vaccine, while 6 cows were used as the unvaccinated controls. All cows were challenged with 1 x 10(5) organisms 4 weeks after the second or the only vaccination. After challenge, cervico-vaginal mucus (CVM) samples were cultured for T . fetus weekly for 9 weeks. Whole cell vaccines were superior to fragmented cell vaccines, and both performed better than no vaccination for apparent elimination of trichomonad infections in dairy cows. In addition, 2V was superior to 1V, which, in turn, was superior to no vaccination. Furthermore, clearance time was reduced most by 2V and whole cell vaccination compared with 1V and fragmented cell vaccination. Clearance time was decreased significantly in all vaccinated cows compared with that in unvaccinated cows.     

Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors

Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.     

动物细胞系的染色体组型与遗传变异率分析

在建立国内首家犬,猫,猴,鼠传代细胞库,即7种动物肾细胞系（F－81,CRFK,MDCK,Vero,Vero-2,MA-104,BHK-21)的种子库和工作库的基础上,通过细胞染色体组型,G带核型,染色体数目变异率,结构畸变率分析,了解7种细胞系传代培养不同代次的染色体变异情况,以相应的细胞株皮下接种褐 形成肿瘤实验,软琼脂细胞克隆一苦恼经与植物凝集素作用下细胞凝集实验为对照,筛选出无致癌／致瘤性,符合细胞遗传学要求,无传染因子污染的细胞系（F－81,CRFK,Vero,Vero-2)或极低致癌性的MDCK细胞系用于制苗,发现肿瘤细胞系高变异率株可在裸鼠体内快速选育成功,细胞系染色体遗传特征决定致性质并具有种属特异性,得到一些100％成瘤和100％不成瘤的细胞株并了与染色体组型的关系,对于肿瘤的发病机理及实验治疗,都是非常好的模型,一些细胞系不仅成瘤而且还可转移（致恶性横纹肌样瘤的BHK－21和Vero 细胞株）,其他致瘤细胞株只成瘤不转移或不明显转移。     

Toward understanding the different function of two types of parenchyma cells in bamboo culms

The bamboo, woody monocot, has two types of parenchyma cells in the ground tissues of its culm, in contrast to a single type of parenchyma cell in rice, maize and other major crop species. The distribution of cell wall components, including lignin, (1-->3), (1-->4)-beta-D-glucans (MGs), the highly-substituted glucuronoarabinoxylans (hsGAXs) and low-branched xylans (lbXs) in ground parenchyma tissue of Phyllostachys heterocycla var. pubescens culms was studied at various developmental stages using light microscopy (LM), UV-microscopy, transmission electron microscopy (TEM) and immunolabeling techniques. The short parenchyma cell walls were lignified in 2-month-old bamboo culms just as the long parenchyma cell walls were. The lignified regions were confined to the portions in contact with the long parenchyma cell walls, while the walls at the cell corner region never lignified, even in 7-year-old culms. Significant differences were also found in the hemicellulose distribution between the short and long parenchyma cell walls. In bamboo parenchyma tissue, MGs were localized in short parenchyma cell walls and few were found in long parenchyma cell walls in both young and 7-year-old culms. The distribution of hsGAXs was similar to that of MGs in young culms, but they only appeared in the cell corner region of short parenchyma cells in old culms. Low-branched xylans were distributed in the lignified, but not in unlignified parenchyma cell walls. Based on this evidence, the differences of function in both short and long parenchyma cells in a bamboo culm are discussed.     

Comparison of endogenous TP53 genomic status with clonogenicity and different modes of cell death after X irradiation

Although extensive data indicate that the tumor suppressor TP53 modifies the radiation responses of human and rodent cells, the exact relationship between TP53 and radiation responsiveness remains controversial. To elucidate the relevance of endogenous TP53 genomic status to radiosensitivity in a cell-type-independent manner, different cells of 10 human tumor cell lines with different tissues of origin were examined for TP53 status. The TP53 status was compared with radiation-related cell survival parameters (D(q), D(0), SF2) and with the mode of cell death. Different modes of cell death were examined by measuring radiation-induced micronucleation, apoptosis and abnormal cells. Alterations of the TP53 gene were detected in eight cell lines. No splicing mutation was found. Five cell lines showed codon 68 polymorphism. Codon 72 alterations were found in four cell lines. "Hot spot" alterations were detected in only two of 10 cell lines. Although the cells differed widely in survival parameters (D(q), D(0), SF2) and modes of cell death (micronucleation/apoptosis/abnormal cells) after irradiation, significant cell-type-independent correlations were obtained between the multiple cell death parameter micronucleation/apoptosis/abnormal cells and SF2 (P < 0.001) and D(q) (P = 0.003). Moreover, cells with a wild-type TP53 gene were more resistant to X rays than cells with a mutated TP53 gene or cells that were TP53-deficient. The alterations within exons 5-10 of the TP53 correlated with a enhanced radiosensitivity. For the first time, we demonstrated a correlation between endogenous genetic alterations within exons 5-10 of TP53 and radiation-related cell survival and cell death. This indicates a new molecular relevance of TP53 status to intrinsic cellular radiosensitivity.     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

The characterization of SV40-transformed cell lines derived from mouse teratocarcinoma: growth properties and differentiated characteristics.

Mouse teratocarcinoma cells derived from embryoid bodies of 129SVsl mice were cultured in vitro to permit their differentiation. These cells were then infected with simiam virus 40 (SV40) and 31 cloned cell lines (SVTER) were derived from these cultures. All 31 SVTER cell lines contained the SV40 tumor (T) antigen and grew as permanent lines in culture. Mock-infected embryoid body cultures did not give rise to permanent cell lines. The morphology of each SVTER cell line was distinct and did not change during successive subclonings. The growth properties and tumorigenic potential of all 31 SVTER cell lines were investigated. None of these lines produced tumors in 129SVsl mice. Each cell line was tested for its ability to (1) grow in medium containing 1% serum, (2) plate on cell monolayer, and (3) form clones in methocel suspension. Only three of the SVTER cell lines were transformed with respect to all three of these criteria. Most of these cell lines were minimal transformants. The SVTER cell lines were tested for creatine phospholinase (CPK), an enzyme activity chracteristic of mouse brain and muscle tissue, and the protease, plasminogen activator (PA) which is found in embryoid bodies and several differentiated cell types. Some of the SVTER cell lines contained high levels of CPK, while others had high levels of PA and a third group of cells contained neither enzyme activity. No SVTER cell line was found with high levels of both these enzyme activities. This result suggests that mutually exclusive sets of genes are expressed in these cells as might be expected from the distinct tissue distribution of the two enzyme activities studied. These SVTER cell lines may be useful in reconstructing developmental pathways of differentiating teratomas in vitro.     

Pectic epitopes are differentially distributed in the cell walls of potato (Solanum tuberosum) tubers

Six monoclonal antibodies (mAbs) were used to map the distribution of pectic epitopes in the cell walls of potato ( Solanum tuberosum L. cvs Kardal and Karnico) tuber tissue in both light and electron microscopes. Unesterified (mAb JIM 5 epitope) and methyl-esterified (mAb JIM 7 epitope) pectins were abundant and equally distributed in all parenchymal and vascular cell walls. Homogalacturonans (HGAs) involved in Ca²⁺-cross-linking (mAb 2F4 epitope) were localised to the middle lamella and abundant at cell corners. The tuber cortex was densely labelled, but parenchymal cell walls in the perimedullary region contained few epitopes of calcium pectate except at corners and pit fields. In contrast, pectic side-chains were not detectable in the middle lamella of all parenchymal cell walls, except in the cortex where mAb LM6 (arabinan epitope) labelled the entire wall. The galactan epitope (mAb LM5) was localised to a zone very close to the plasmalemma in cortical cell walls and was also less abundant at pit fields and in vascular cell walls. MAb CCRC-M2 (rhamnogalacturonan I epitope) did not cross-react. Our results show that the cell walls of potato tubers are not homogeneous structures and that the pectic composition of the walls is spatially regulated.     

Characterization of porous PLGA/PLA microparticles as a scaffold for three dimensional growth of breast cancer cells

We have designed and evaluated biodegradable porous polymeric microparticles as a scaffold for cell growth. The hypothesis was that microparticles with optimized composition and properties would have better cell adhesion and hence cell growth into a tissue-like structure. Solvent-evaporation method was modified using sucrose as an additive to form large porous microparticles of poly(D,L-lactic-co-glycolic) (PLGA) and polylactide (PLA) polymers. Microparticles containing hydrophilic polymers (poly(vinyl alcohol) and chitosan) incorporated in their internal matrix structure were also formulated. Different formulations of microparticles were evaluated for physical properties, cell adhesion, and cell growth in culture. PLA microparticles containing poly(vinyl alcohol) (PVA) in the matrix structure (PLA-PVA) and treated with serum prior to cell seeding demonstrated better cell adhesion and cell growth than other formulations of microparticles. Cells were seen to grow into clumps, engulfing microparticles completely with time, and forming a 3-D tissue-like structure. Cell density of 1.5 x 10(6) cells per mg of microparticles was achieved in 9 days of culture, which was a 7-fold increase from the initial seeding cell density. The mechanism of better cell growth on PLA-PVA microparticles appears to be due to the PVA associated with the internal matrix structure of microparticles. These microparticles demonstrated better wetting in culture and also cell adhesion. In addition to tissue engineering applications, microparticles with cancer cells grown into a tissue-like structure in vitro can be potentially used as a model system for preclinical evaluation of the cytotoxic effect of anticancer agents.     

Bcl-x(L) mediates increased production of humanized monoclonal antibodies in Chinese hamster ovary cells

Enhanced product yields, reduction in throughput time, improved cost-effectiveness and product quality are examples of benefits gained by delaying apoptotic cell death in bioreactors. To examine the effect on recombinant protein production, bcl-x(L) was overexpressed in a CHO cell line secreting humanized monoclonal antibody directed against the alpha1beta1 integrin. When cell lines overexpressing bcl-x(L) were compared to the parent, cell viability was increased by 20% and titers by 80%. Total viable cell densities were similar and specific productivities were enhanced by almost two-fold on scale-up to bioreactors. Comparison in a chemically defined media demonstrated an even greater sustained viability in bcl-x(L) expressing cells by 50% and up to 90% increase in titer with no impact on product quality. Caspase 3 activities were monitored as a marker for apoptotic cell death. In the presence of Bcl-x(L), caspase activities were reduced to background levels. The role of Bcl-x(L) in protecting cells from premature death was further examined in studies performed in the presence of NaBu, at concentrations known to trigger cell death. Results demonstrated that cells expressing bcl-x(L) retained 88% cell viability with >2 fold increase in titer. Bcl-x(L) was similarly overexpressed in a different CHO cell line producing a humanized mAb against the chemokine MCP1. Once again, production titer was increased by 80% and viability by 75%. Together the studies have shown that overexpression of bcl-x(L) in production cell lines was able to significantly increase the titer by enhancing both the specific activity and total cell viability while maintaining product quality.