A recombinant bacteriophage-based assay for the discriminative detection of culturable and viable but nonculturable Escherichia coli O157:H7

A previously green fluorescent protein (GFP)-labeled PP01 virulent bacteriophage, specific to Escherichia coli O157:H7, was used to construct lysozyme-inactivated GFP-labeled PP01 phage (PP01e-/GFP). The new recombinant phage lacked lytic activity because of the inactivation of gene e, which produces the lysozyme responsible for cell lysis. Gene e was inactivated by inserting an amber stop codon. Prolonged incubation of E. coli O157:H7 cells with PP01e-/GFP did not lead to cell lysis, while the propagation of PP01e-/GFP in host cells increased the intensity of green fluorescence. Retention of cell morphology and increase in fluorescence enabled the direct visualization and enumeration of E. coli O157:H7 cells within an hour. The PP01e-/GFP system, when combined with nutrient uptake analysis, further allowed the discriminative detection of culturable, viable but nonculturable (VBNC), and dead cells in the stress-induced aquatic environment. Stress-induced cells, which retained culturability, allowed phage propagation and produced bright green florescence. Nonculturable cells (VBNC and dead) allowed only phage adsorption but no proliferation and remained low fluorescent. The low-fluorescent nonculturable cells were further differentiated into VBNC and dead cells on the basis of nutrient uptake analysis. The low-fluorescent cells, which grew in size by nutrient incorporation during prolonged incubation in nutrient medium, were defined as metabolically active and in the VBNC state. The elongated VBNC cells were then easily recognizable from dead cells. The proposed assay enabled the detection and quantification of VBNC cells. Additionally, it revealed the proportion of culturable to VBNC cells within the population, as opposed to conventional techniques, which demonstrate VBNC cells as a differential value of the total viable count and the culturable cell count.     

Limits of propidium iodide as a cell viability indicator for environmental bacteria.

BACKGROUND: Viability measurements of individual bacteria are applied in various scopes of research and industry using approaches where propidium iodide (PI) serves as dead cell indicator. The reliability of PI uptake as a cell viability indicator for dead (PI permeable) and viable (PI impermeable) bacteria was tested using two soil bacteria, the gram(-) Sphingomonas sp. LB126 and the gram(+) Mycobacterium frederiksbergense LB501T. METHODS: Bacterial proliferation activities observed viaDAPI and Hoechst 33342 staining were linked to the energy charge and the proportion of dead cells as obtained by diOC(6) (3)-staining and PI-uptake, respectively. Calibration and verification experiments were performed using batch cultures grown on different substrates. RESULTS: PI uptake depended on the physiological state of the bacterial cells. Unexpectedly, up to 40% of both strains were stained by PI during early exponential growth on glucose when compared to 2-5% of cells in the early stationary phase of growth. CONCLUSIONS: The results question the utility of PI as a universal indicator for the viability of (environmental) bacteria. It rather appears that in addition to nonviable cells, PI also stains growing cells of Sphingomonas sp. and M. frederiksbergense during a short period of their life cycle.     

Affinity of single S. cerevisiae cells to 2-NBDglucose under changing substrate concentrations.

BACKGROUND: Saccharomyces cerevisiae is a widely employed microorganism in biotechnological processes. Since proliferation and product formation depend on the capacity of the cell to access and metabolize a carbon source, a technique was developed to enable for analyzing the S. cerevisiae H155 cells' affinity to extracellular glucose concentrations. METHODS: The fluorescent glucose analogue 2-NBDglucose was employed as a functional parameter to analyze the cells' affinity to glucose. Structural parameters (proliferation, neutral lipid content, granularity, and cell size) were also investigated. Cells were grown both in batches and in chemostat regimes. RESULTS: The 2-NBDglucose uptake in individual cells proceeds in a time- and concentration-dependent manner and is affected by respiratory and respirofermentative modes of growth. The process is inhibited by D-glucose, D-fructose, D-mannose, and sucrose, but not L-glucose, D-galactose or lactose; maltose is a weak inhibitor. The affinity of the individual cells to 2-NBDglucose was found to be high at low extracellular glucose concentrations, and weak at high concentrations. An additional, underlying pattern in the cells' affinity to glucose was detected, illustrated by the recurrent appearance of two subpopulations showing distinctly differing quantities of this substrate. CONCLUSIONS: A multiparameter flow cytometry approach is presented that enables, for the first time, for analysis of the affinity of individual S. cerevisiae cells to glucose. Besides the adjustment of the yeast cell metabolism to extracellular glucose concentrations by altering their affinity to glucose, at least one further mechanism is clearly involved. Two subpopulations of cells were resolved, with different affinities not correlated with other cellular parameters measured.     

Effect of cultivation conditions on the growth properties of Swiss 3T3 cells

A possibility of using Swiss 3T3 cells, adapted to the growth in the Eagle basal medium and bovine serum, in studies of cell proliferation and quiescent state was shown on the basis of their growth characteristics. Proliferative activity of cultures was estimated by measuring the intensity of DNA synthesis (incorporation of labeled thymidine and flow cytofluorometric analysis), mitotic index and cell number counts. Growth rate and saturation density of the culture were analyzed depending on serum concentration, substrate quality and medium changes both in growth and quiescent states. In spite of repeated medium changes such adapted cells had saturation density within 4.10(4)--7.10(4) cells/cm2, standard for this line. Besides, a distinct inhibition of cell proliferation at confluence or after incubation with low serum (0.5%) and a possibility of the following stimulation of cell divisions by adding a fresh medium containing different concentrations of serum were demonstrated. The increased rate of adipose conversion was detected in resting confluent 3T3 cells cultivated in closed vessels, as compared to cells growing in tissue culture dishes in the CO2 incubator.     

An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations.

BACKGROUND: When using traditional microbiological techniques to monitor cell proliferation and viability, stressed, sublethally injured, or otherwise "viable but nonculturable" cells often go undetected. Because of this, such cells often are not considered by mathematical models used to predict bioprocess performance on scale-up and inaccuracies result. Therefore, analytical techniques, decoupled from postsampling growth, are desirable to rapidly monitor individual cell physiologic states during microbial fermentations. METHODS: Microbial cells, including Escherichia coli, Rhodococus sp., and Sacharomyces cerevisiae, were taken at various stages from a range of fermentation processes and stained with one of three mixtures of fluorescent stains: rhodamine 123/propidium iodide, bis-oxonol/propidium iodide, or bis-oxonol/ethidium bromide/propidium iodide. An individual cell's physiologic state was assessed with a Coulter Epics Elite analyzer based on the differential uptakes of these fluorescent stains. RESULTS: It was possible to resolve an individual cell's physiologic state beyond culturability based on the functionality of dye extrusion pumps and the presence or absence of an intact polarized cytoplasmic membrane, enabling assessment of population heterogeneity. This approach allows the simultaneous differentiation of at least four functional subpopulations in microbial populations. CONCLUSIONS: Fluorescent staining methods used in our laboratories have led to a functional classification of the physiological state of individual microbial cells based on reproductive activity, metabolic activity, and membrane integrity. We have used these techniques extensively for monitoring the stress responses of microorganisms in such diverse areas as bioremediation, biotransformation, food processing, and microbial fermentation; microbial fermentation is discussed in this article.     

Transport characteristics of peptides and peptidomimetics: II. Hydroxyethylamine bioisostere-containing peptidomimetics as substrates for the oligopeptide transporter and P-glycoprotein in the intestinal mucosa.

Peptide bond bioisosteres, such as hydroxyethylamine (Hea), have frequently been used to stabilize metabolically labile peptide bonds in peptidomimetic drug design in an effort to increase the oral bioavailability of drug candidates. However, the impact of the peptide bond bioisosteres on the cell permeation characteristics of peptidomimetics is not well understood, particularly with respect to the effects on the substrate activity for proteins that can restrict (e.g. P-glycoprotein, P-gp) or facilitate (e.g. the oligopeptide transporter, OPT) intestinal mucosal permeation of peptidomimetics. In this study, terminally free and terminally modified (N-acetylated and C-amidated) peptidomimetics of H-Ala-Phe-OH and H-Ala-Phe-Ala-OH with the Ala-Phe peptide bonds replaced by Hea bioisosteres were synthesized. Transport characteristics of these peptidomimetics were investigated using Caco-2 cell monolayers as an in vitro model of the intestinal mucosa. The study showed that the Hea bioisostere stabilized the peptidomimetics to protease metabolism in Caco-2 cells. All terminally free peptidomimetics showed significant affinity and substrate activity for OPT. The affinity and substrate activity for OPT were stereoselective for peptidomimetics containing an S,S-configuration for the two adjacent chiral centers related to the Hea bioisostere. Three of the four terminally modified peptidomimetics showed significant substrate activity for P-gp and, interestingly, the substrate activity for P-gp was also stereoselective; however, it was in favor of an R,R-configuration for the two adjacent chiral centers related to the Hea bioisostere.     

DAPI染色法在流式细胞术中去除死细胞影响的探讨

目的:探索利用DAPI染色法在流式细胞检测过程中去除死细胞,消除死细胞对流式结果的影响。方法:建立原位肝癌小鼠模型,流式检测其中死细胞对脾脏中骨髓来源抑制性细胞(myeloid-derived suppressor cell,MDSC)结果的影响,并以碘化丙啶(propidiumiodide,PI)为对照,探索不同DAPI浓度、不同染色时间对死细胞比例的影响。结果:流式细胞术中去除死细胞影响时DAPI染色浓度低于1 g/m L时,其染色结果影响不大,浓度大于1 g/m L,死细胞比例明显升高;当DAPI使用浓度为0.2 g/m L时,在30钟内死细胞染色结果与PI相比差别无统计学差异,当染色时间大于30 min时,死细胞的比例显著升高。结论:DAPI染色法应用于流式细胞术中去除死细胞是一种可靠的检测方法,且简单易行,推荐使用方法为0.2 g/m L,染色时间1-5 min。     

Interaction between catalytically inactive calpain and calpastatin. Evidence for its occurrence in stimulated cells

Conformational changes in the calpain molecule following interaction with natural ligands can be monitored by the binding of a specific monoclonal antibody directed against the catalytic domain of the protease. None of these conformational states showed catalytic activity and probably represent intermediate forms preceding the active enzyme state. In its native inactive conformation, calpain shows very low affinity for this monoclonal antibody, whereas, on binding to the ligands Ca(2+), substrate or calpastatin, the affinity increases up to 10-fold, with calpastatin being the most effective. This methodology was also used to show that calpain undergoes similar conformational changes in intact cells exposed to stimuli that induce either a rise in intracellular [Ca(2+)] or extensive diffusion of calpastatin into the cytosol without affecting Ca(2+) homeostasis. The fact that the changes in the calpain state are also observed under the latter conditions indicates that calpastatin availability in the cytosol is the triggering event for calpain-calpastatin interaction, which is presumably involved in the control of the extent of calpain activation through translocation to specific sites of action.     

Conformational states of the corticotropin releasing factor 1 (CRF1) receptor: detection, and pharmacological evaluation by peptide ligands

Previous corticotropin releasing factor 1 (CRF₁) receptor characterization has been performed using radiolabeled agonists, which bind predominantly the receptor-G-protein complex. The pharmacological profile of other receptor states, and their abundance, remain poorly characterized. Here we investigated the affinity states of the CRF₁ receptor heterologously expressed in Ltk⁻ cells and endogenously expressed in rat cerebellum. In L-CRF₁ cell membranes, three agonist affinity states were detected: a very-high affinity receptor-G-protein complex state (eliminated by GTPγS) bound by [¹²⁵I]sauvagine (43 pM, RG); a high affinity state insensitive to GTPγS bound by [¹²⁵I]sauvagine (1.4 nM, termed R_O); and a low affinity G-protein-uncoupled state detected by sauvagine displacement of [¹²⁵I]astressin, a labeled antagonist (120 nM, R). The relative abundance of RG:R_O:R was 18%:16%:66%. All three states were demonstrated in rat cerebellum with similar relative abundance (15%:16%:69%). The R state bound CRF with low affinity (270–330 nM), displayed a novel rank order of ligand affinity, and represented the majority of the receptor population in both receptor preparations. This study provides a framework to identify CRF₁ receptor conformational states in various receptor preparations.     

抑制糖酵解活性限制雄性原始生殖细胞的增殖

目的:雄性原始生殖细胞在植入生殖嵴后,会从有丝分裂退出进入静息状态,在这一过程中伴随着细胞内代谢状态的改变,本研究旨在体解析原始生殖细胞增殖的改变与细胞代谢之间的因果关系。方法:通过体内Brdu掺入实验明确不同时间点雄性生殖细胞的增殖状态;分析比较增殖状态和静息状态原始生殖细胞糖酵解相关基因的表达;利用腹腔注射HK2特异性抑制剂2-Deoxy-D-glucose (2-DG),构建糖酵解抑制小鼠模型;通过免疫荧光与qPCR分析抑制糖酵解后原始生殖细胞的表型。结果:免疫荧光结果显示雄性生殖细胞增殖停滞从E13.5开始,至E15.5完全停滞;qPCR和Western Blot显示在此过程中HK2的表达是逐渐降低的;在E11.5抑制小鼠胚胎中的糖酵解过程,可以在E13.5检测到雄性PGCs增殖下降,并且可以抑制多能性基因如Sox2、Oct4的表达。结论:研究发现,E11.5-E13.5雄性原始生殖细胞内增殖与多能性的维持需要糖酵解。改变胚胎糖酵解水平可以影响原始生殖细胞增殖分化进程。     

Pitfalls of cell-systematic evolution of ligands by exponential enrichment (SELEX): existing dead cells during in vitro selection anticipate the enrichment of specific aptamers

Aptamers represent auspicious ligands for recognition of target molecules on the surface of a specific cell population, such as stem or cancer cells. These ligands are able to capture and enrich desired cells from a cell mixture, and can be used for identification of new biomarkers, development of cell-specific therapeutics, and stem cell therapy. In this study, we investigated the influence of dead cells on single-stranded DNA (ssDNA) binding and established a method to eliminate dead cells from a cell suspension. Flow cytometry analyses demonstrated that all dead cells were stained with fluorescein-labeled ssDNA molecules. The increasing of the proportion of dead cells led to an increased number of cells that were positive for ssDNA staining. Using dead cell removal microbeads, the proportion of dead cells was significantly reduced. The studies demonstrated that dead cells lead to unspecific uptake/binding of ssDNA molecules during cell-Systematic Evolution of Ligands by Exponential enrichment (SELEX) and can cause failure of the selection process. Thus, the elimination of dead cell population before incubation with ssDNA molecules will reduce the loss of target binding sequences and the contamination of the enriched aptamer pool with unspecific ssDNA molecules caused by unspecific binding to dead cells.     

基于生物阻抗谱的生物细胞活性免标记检测方法

目的 基于生物阻抗谱（bioelectrical impedance spectroscopy,BIS）技术,提出一种免标记的生物细胞活性实时检测方法。该方法依据不同浓度、生理、病理状态下细胞组织的电学特性差异来判断细胞是否具有活性,以协助医师在临床手术中快速精准定位患者烫伤组织并实现有效切除。方法 使用具有活性的斑马鱼胚胎干细胞来模拟人体烫伤组织,采用生物阻抗谱技术来鉴别细胞组织的生理状态。结果 在不同状态下,细胞的阻抗幅值变化有显著的差异,可以从中发现同等浓度下活性细胞的阻抗幅值比死亡细胞平均高出17.25%,活性细胞发生弛豫频率的时间也比死亡细胞早25%。结论 实验数据表明,生物阻抗谱法能有效区分胚胎干细胞的两类生理状态;从聚类区域中可以看出,BIS检测法具有明显的细胞活性及浓度区分能力,理论上能够快速地协助医师完成对患者烫伤组织检测。     

In vitro effects of microwave radiation on rat liver mitochondria

Liver mitochondria were exposed in vitro at 30°C to microwave radiation (2.45 GHz) during the following states of respiraton: resting, state 1; substrate dependent, state 2; ADP stimulated, state 3; and ADP depleted, state 4. At 10 or 100 mW/g, with succinate as substrate, no effect of exposure was observed on states 1–4 or the respiratory control index (state 3/state 4) of either tightly or loosely coupled mitochondria. When glutamate was used as substrate, no effects were observed at 10 mW/g. However, in the loosely coupled mitochondria the 100 mW/g exposure produced an increase in states 2 and 4 and a decrease in the respiratory control index. The results suggest that the function of loosely coupled mitochondria can be affected at high power levels of microwave radiation.     

Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.     

Potentiation of endothelial cell proliferation by fibrin(ogen)-bound fibroblast growth factor-2.

Endothelial cell growth is stimulated by fibroblast growth factor-2 (FGF-2), and both adhesion and proliferation are modulated by interactions with fibrinogen and fibrin. Previous evidence indicates that FGF-2 binds specifically and with high affinity to fibrinogen and fibrin, suggesting that their effects on endothelial cells may be coordinated. In this study, we have, therefore, investigated the ability of FGF-2 bound to fibrinogen and fibrin to stimulate proliferation of endothelial cells. Human umbilical vein endothelial cells were cultured in the presence of FGF-2 with or without fibrinogen, and proliferation was assessed by microscopic examination of cultures, incorporation of [3H]thymidine and by cell counting. Cells cultured in the presence of both FGF-2 and fibrinogen proliferated more rapidly than those with FGF-2 alone and exhibited a decreased population doubling time. At concentrations of FGF-2 up to 150 ng/ml, there was greater endothelial cell proliferation in the presence of fibrinogen than in its absence with the most pronounced effect below 1 ng/ml. The maximum effect of fibrinogen was observed at a molar ratio of fibrinogen to FGF-2 of 2:1, corresponding to the maximum molar binding ratio. Endothelial cells proliferated when plated on fibrin or surface-immobilized fibrinogen with FGF-2, indicating that FGF-2 bound to surface-associated fibrin(ogen) retained activity. We conclude that fibrinogen- or fibrin-bound FGF-2 is able to support endothelial cell proliferation and that fibrinogen potentiates the proliferative capacity of FGF-2.     

孕激素诱导cyclin G1在小鼠子宫内膜上皮细胞的表达及其对细胞增殖的影响

本文在体外培养条件下研究卵巢激素诱导小鼠子宫内膜上皮细胞cyclin G1的表达及细胞增殖和细胞周期进程的变化,以探讨孕激素依赖的细胞周期调控因子cyclin G1对子宫内膜上皮细胞增殖的负调控作用.原代培养小鼠子宫内膜上皮细胞,待其生长汇合后分为4组:对照组(C组)、雌激素组(E组)、孕激素组(P组)、雌、孕激素共同作用组(EP组).加入相应激素作用24 h后,用细胞免疫化学方法检测各组细胞cyclin G1的表达水平:四甲基偶氮唑蓝(MTT)比色法检测各组细胞活力,间接观察子宫内膜上皮细胞的增殖情况;用流式细胞仪检测分布在细胞周期各时相的子宫内膜上皮细胞所占百分数.细胞免疫化学结果显示,cyclin G1在C组和E组子宫内膜上皮细胞上无明显表达,而在P组和EP组子宫内膜上皮细胞中表达明显,且定位于细胞核内.MTT法结果显示,与C组相比,E组细胞活力明显增高,而P组和EP组的细胞活力均明显下降,表明雌激素能促进子宫内膜上皮细胞增殖,而孕激素则具有抑制子宫内膜上皮细胞增殖的作用.流式细胞术检测显示,与C组相比,E组中处于S期的子宫内膜上皮细胞百分数增多;P组与EP组中处于S期的子宫内膜上皮细胞百分数明显减少,而处于G1期的细胞百分数和G2/M期的细胞百分数则明显增加.上述结果提示,孕激素依赖的cyclin G1可能通过阻滞细胞周期进程来参与孕激素对子宫内膜上皮细胞增殖的负调控作用.     

Influence of pulsed electromagnetic and pulsed vector magnetic potential field on the growth of tumor cells

Aims and Background: Tumor diseases cause 20% of deaths in Europe and they are the second most common cause of death and morbidity after cardiovascular diseases. Thus, tumor cells are target of many therapeutic strategies and tumor research is focused on searching more efficient and specific drugs as well as new therapeutic approaches. One of the areas of tumor research is an issue of external fields. In our work, we tested influence of a pulsed electromagnetic field (PEMF) and a hypothetic field of the pulsed vector magnetic potential (PVMP) on the growth of tumor cells; and further the possible growth inhibition effect of the PVMP. Methods: Both unipolar and bipolar PEMF fields of 5?mT and PVMP fields of 0?mT at frequencies of 15?Hz, 125?Hz and 625?Hz were tested on cancer cell lines derived from various types of tumors: CEM/C2 (acute lymphoblastic leukemia), SU-DHL-4 (B-cell lymphoma), COLO-320DM (colorectal adenocarcinoma), MDA-BM-468 (breast adenocarcinoma), and ZR-75-1 (ductal carcinoma). Cell morphology was observed, proliferation activity using WST assay was measured and simultaneous proportion of live, early apoptotic and dead cells was detected using flow cytometry. Results: A PEMF of 125?Hz and 625?Hz for 24?h–48?h increased proliferation activity in the 2 types of cancer cell lines used, i.e. COLO-320DM and ZR-75-1. In contrast, any of employed methods did not confirm a significant inhibitory effect of hypothetic PVMP field on tumor cells.     

Immunoregulatory functions of paf-acether. III. Down-regulation of CD4+ T cells high-affinity IL-2 receptor expression

In the present report, we further explored the mechanisms by which 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (paf-acether), a phospholipid mediator of inflammation inhibited PHA-induced CD4+ cell proliferation. Evidence was obtained that CD4+ cells stimulated with either PHA or immobilized OKT3 in the presence of paf at concentrations that block CD4+ cell proliferation, exhibited a marked decrease in high affinity IL-2R expression. Importantly, paf did not prevent the binding of IL-2 to its receptor. Scatchard analysis of the binding data indicated that paf caused more than 50% decrease in the number of IL-2 high affinity sites per cell, whereas the receptor ligand affinity remained essentially constant. Moreover, the down-regulation of high affinity IL-2R was also accompanied by a loss of IL-2-dependent proliferative capacity. Together these data suggest that decreased expression of high affinity IL-2R may contribute to the diminished proliferative activity observed in CD4+ cells stimulated with PHA or immobilized OKT3 in the presence of paf. They further emphasize the potential role of lipid proinflammatory mediators such as paf in the regulation of T cell activation.     

Isolation and characterization of a macrophage-derived heparin-binding growth factor.

Human mononuclear cells were plated in culture, and the conditioned media of these cells were analyzed by heparin-Sepharose affinity chromatography. The fractions were tested for growth factor activity as measured by the stimulation of DNA synthesis in BALB/c 3T3 cells. After 2 d in culture, two peaks of heparin-binding growth factor (HBGF) activity were detected, one eluting with 0.5 M NaCl, which could be shown to be platelet-derived growth factor (PDGF)-like, and the other eluting with 1.0 M NaCl. After 7-11 d in culture, when monocytes had clearly differentiated into macrophages, greater than 95% of the HBGF activity in conditioned medium consisted of the 1.0 M NaCl elution peak. This activity, which was designated macrophage-derived HBGF (MD-HBGF), was found to be a cationic heat-resistant polypeptide with a molecular weight in the range of 14-25 kDa. Analysis using Western blots and specific neutralizing antisera, as well as comparative heparin affinity analysis, indicated that MD-HBGF was not identical to other heparin-binding 3T3 cell growth factors known to be produced by macrophages, such as PDGF (AB, AA, and BB forms), acidic fibroblast growth factor, and basic fibroblast growth factor. In addition to stimulating mitogenesis in 3T3 cells, MD-HBGF also stimulated the proliferation of vascular smooth muscle cells, but did not stimulate the proliferation of vascular endothelial cells.     

Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry

The viability of the polychlorinated biphenyl-degrading bacterium Comamonas testosteroni TK102 was assessed by flow cytometry (FCM) with the fluorogenic ester Calcein-AM (CAM) and the nucleic acid dye propidium iodide (PI). CAM stained live cells, whereas PI stained dead cells. When double staining with CAM and PI was performed, three physiological states, i.e., live (calcein positive, PI negative), dead (calcein negative, PI positive), and permeabilized (calcein positive, PI positive), were detected. To evaluate the reliability of this double-staining method, suspensions of live and dead cells were mixed in various proportions and analyzed by FCM. The proportion of dead cells measured by FCM directly correlated with the proportion of dead cells in the sample (y = 0.9872 x + 0.18; R(2) = 0.9971). In addition, the proportion of live cells measured by FCM inversely correlated with the proportion of dead cells in the sample (y = -0.9776 x + 98.36; R(2) = 0.9962). The proportion of permeabilized cells was consistently less than 2%. These results indicate that FCM in combination with CAM and PI staining is rapid (相似文献