Molecular phenotyping of aging in single yeast cells using a novel microfluidic device

Budding yeast has served as an important model organism for aging research, and previous genetic studies have led to the discovery of conserved genes/pathways that regulate lifespan across species. However, the molecular causes of aging and death remain elusive, because it is very difficult to directly observe the cellular and molecular events accompanying aging in single yeast cells by the traditional approach based on micromanipulation. We have developed a microfluidic system to track individual mother cells throughout their lifespan, allowing automated lifespan measurement and direct observation of cell cycle dynamics, cell/organelle morphologies, and various molecular markers. We found that aging of the wild-type cells is characterized by an increased general stress and a progressive lengthening of the cell cycle for the last few cell divisions; these features are much less apparent in the long-lived FOB1 deletion mutant. Following the fate of individual cells revealed that there are different forms of cell death that are characterized by different terminal cell morphologies, and associated with different levels of stress and lifespan. We have identified a molecular marker - the level of the expression of Hsp104, as a good predictor for the lifespan of individual cells. Our approach allows detailed molecular phenotyping of single cells in the process of aging and thus provides new insight into its mechanism.     

Dynamic functional and structural analysis of living cells: New tools for vital staining of nuclear DNA and for characterisation of cell motion

Increasing interest has been paid to applications of fluorescence measurements to analyze physiological mechanisms in living cells. However, few studies have taken advantage of DNA quantification by fluorometry for dynamic assessment of chromatin organization as well as cell motion during the cell cycle. This approach involves both optimal conditions for DNA staining and cell tracking methods. In this context, this report describes a stoichiometric method for nuclear DNA specific staining, using the bisbenzimidazole dye Hoechst 33342 associated with verapamil, a calcium membrane channel blocker. This method makes it possible to correlate variations of nuclear DNA content with cell motion in cells that are maintained alive. Motion measurement is the second goal of this paper and it explains the snake-spline method, and the associated cell following method.     

Comparative physiology and molecular analysis of carbonic anhydrase from the red blood cells of teleost fish

This study investigates the early evolution of vertebrate red blood cell (rbc) carbonic anhydrase (CA) by examining the physiological and molecular properties of rbc CA in teleost fish. When representatives of four different families of teleosts were compared, it was found that differences in overall rbc CA activity were due to different concentrations of CA, rather than differences in the enzymes kinetic properties. Additional molecular analysis of CA from the rbcs of rainbow trout provided further evidence that critical elements of the enzyme, such as the active site, have been highly conserved during vertebrate evolution. The active site of the trout CA differed from that of gar rbc CA at only two amino acid positions. The rainbow trout rbc CA sequence also showed high sequence homology with CA sequences from other fish tissues, and fits into an emerging group of fish CAs that are basal to mammalian CA I, II and III. Northern blot analysis of the tissue expression of the sequenced CA indicated that it is primarily found in the rbcs, but high amounts of cytosolic CA activity were also found in the gill, suggesting the presence of other cytosolic CA isozymes in this species.Abbreviations Az acetazolamide - CA carbonic anhydrase - MP maximum parsimony - NJ neighbour joining - RACE rapid amplification of cDNA ends - rbc red blood cellCommunicated by L.C.-H. Wang     

A simple method for the quantitative evaluation of functional effects of xenobiotics with cultured cells

We present a simple, noninvasive, nondestructive all-purpose method for the quantitative evaluation of functional effects of xenobiotics with cultured cells and the work station for its routine, easy implementation. At present 1 to 150 cells growing in one to six dishes can be studied in parallel or otherwise at time intervals ranging from 10 s to 6 h or more, over periods of time ranging from a few tens of minutes to 3–4 days. Any aspect of cell physiological behavior can be studied (differentiation-dedifferentiation, migration, division, degeneration, death) without preliminary staining and/or fixation provided it results in optically visible changes.     

Emerging tools for real-time label-free detection of interactions on functional protein microarrays

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.     

Advanced phenotyping offers opportunities for improved breeding of forage and turf species

Background and Aims

Advanced phenotyping, i.e. the application of automated, high-throughput methods to characterize plant architecture and performance, has the potential to accelerate breeding progress but is far from being routinely used in current breeding approaches. In forage and turf improvement programmes, in particular, where breeding populations and cultivars are characterized by high genetic diversity and substantial genotype × environment interactions, precise and efficient phenotyping is essential to meet future challenges imposed by climate change, growing demand and declining resources.

Scope

This review highlights recent achievements in the establishment of phenotyping tools and platforms. Some of these tools have originally been established in remote sensing, some in precision agriculture, while others are laboratory-based imaging procedures. They quantify plant colour, spectral reflection, chlorophyll-fluorescence, temperature and other properties, from which traits such as biomass, architecture, photosynthetic efficiency, stomatal aperture or stress resistance can be derived. Applications of these methods in the context of forage and turf breeding are discussed.

Conclusions

Progress in cutting-edge molecular breeding tools is beginning to be matched by progress in automated non-destructive imaging methods. Joint application of precise phenotyping machinery and molecular tools in optimized breeding schemes will improve forage and turf breeding in the near future and will thereby contribute to amended performance of managed grassland agroecosystems.     

Emerging functional roles for the glycosyl-phosphatidylinositol membrane protein anchor

Conclusion Experimental evidence has accumulated over the past few years to suggest that the GPI protein anchor may play a broad role in the regulation of membrane protein function. The significant changes in the biophysical properties of proteins that are membrane-anchored through GPI in lieu of a hydrophobic transmembrane peptide indicates a variety phobic transmembrane peptide indicates a variety of potential new functions served by the anchor structure itself. Moreover, the number of structural variations within the family of GPI molecules indicates a further opportunity for subspecialization of such anchored proteins, especially regarding cellular localization, mobility, metabolism and susceptibility to enzymatically-induced release. It is likely that further exploration of the structure and function of the GPI anchor may reveal additional roles for this unusual mechanism of membrane-protein attachment.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Development of an 8-color antibody panel for functional phenotyping of human CD8+ cytotoxic T cells from peripheral blood mononuclear cells

The study of CD8 positive cells in peripheral blood has become an essential part of research in the field of cancer immunotherapies, vaccine development, inflammation, autoimmune disease, etc. In this study, an 8-color flow cytometry panel, containing lineage and functional markers, was developed for the identification of CD8+ cytotoxic T cells in previously cryopreserved peripheral blood mononuclear cells from healthy human donors. By studying functional markers in naïve and CD3/CD28 activated T cells we demonstrate that the panel is capable of detecting protein markers corresponding to different T cell activation statuses. Data generated by flow cytometry were corroborated by different antibody based assay technologies to detect soluble cytokines. Our findings suggest that there is an inter donor variability in both baseline and activation responses. We have also successfully developed an antibody panel for flow cytometry that could be used to study cytotoxic function of CD8 T cells in clinical immunology research areas.     

Thermodynamic studies on oxygen binding by human red blood cells

Oxygen equilibrium curves have been measured on human normal red blood cells, at the temperatures of 20, 25, 30, 37 and 41 degrees C, and at pHs ranging from 6.8 to 8.2. The thermodynamical parameters have been determined for the four successive steps of oxygenation and for overall oxygenation, according to the Adair and MWC models [Monod J, Wyman J, Changeux JP. On the nature of allosteric transitions: a plausible model. J Mol Biol 1965;12:88-118]. The heat release appears to be nearly equal for the four steps. At the first three steps, the delta H change is counterbalanced by a nearly equivalent change of delta S, resulting in a rather small delta G value. delta G is greater at the fourth step, because of diminution of this enthalpy-entropy compensation phenomenon. The four steps are both enthalpy and entropy driven. According to the MWC model, the T to R transition is endothermic, and allosteric quaternary transition occurs at binding of the third oxygen. The average heat release increases by 2.8 kcal/mol when pH raises from 7.4 to 8.2, but flattens below pH 7.4. After correction for the heat of solution of oxygen and for the heat of proton release (referred to intracellular pH), an intrinsic heat for oxygenation of the heme of approximately--13 kcal/mol is obtained for the successive steps of oxygenation (at pH 7.4, 37 degrees C). These results are compared with those previously obtained for pigeon and trout red blood cells.     

Computer tools for the analysis of schooling

Synopsis Quantification of fish school structure is difficult because of: (1) the large amount of positional data that must be recorded, (2) the fact that schools are moving, and (3) the fact that schools are threedimensional. Computer tools for addressing these problems include x,y digitizing devices interfaced to computers and interactive computer graphics systems. General computer algorithms now exist for 3-D reconstruction of schools given any two 2-D views such as photographs.Our computerized film analyzer, the Galatea system, is described in detail. This system allows for rapid, accurate input of multiple points which can be followed through each frame in a film sequence. Its software packages perform 3-D reconstructions, analyze undulatory motion, and do extensive statistical tests.     

Featured Article: Dexamethasone and rosiglitazone are sufficient and necessary for producing functional adipocytes from mesenchymal stem cells

The final product of adipogenesis is a functional adipocyte. This mature cell acquires the necessary machinery for lipid metabolism, loses its proliferation potential, increases its insulin sensitivity, and secretes adipokines. Multipotent mesechymal stromal cells have been recognized as a source of adipocytes both in vivo and in vitro. The in vitro adipogenic differentiation of human MSC (hMSC) has been induced up to now by using a complex stimulus which includes dexamethasone, 3-isobutyl-1-methylxanthine, indomethacin, and insulin (a classical cocktail) and evaluated according to morphological changes. The present work was aimed at demonstrating that the simultaneous activation of dexamethasone’s canonical signaling pathways, through the glucocorticoid receptor and CCAAT-enhancer-binding proteins (C/EBPs) and rosiglitazone through peroxisome proliferator-activated receptor gamma (PPAR-gamma) is sufficient yet necessary for inducing hMSC adipogenic differentiation. It was also ascertained that hMSC exposed just to dexamethasone and rosiglitazone (D&R) differentiated into cells which accumulated neutral lipid droplets, expressed C/EBP-alpha, PPAR-gamma, aP2, lipoprotein lipase, acyl-CoA synthetase, phosphoenolpyruvate carboxykinase, adiponectin, and leptin genes but did not proliferate. Glucose uptake was dose dependent on insulin stimulus and high levels of adipokines were secreted (i.e. displaying not only the morphology but also expressing mature adipocytes’ specific genes and functional characteristics). This work has demonstrated that (i) the activating C/EBPs and PPAR-gamma signaling pathways were sufficient to induce adipogenic differentiation from hMSC, (ii) D&R producing functional adipocytes from hMSC, (iii) D&R induce adipogenic differentiation from mammalian MSC (including those which are refractory to classical adipogenic differentiation stimuli). D&R would thus seem to be a useful tool for MSC characterization, studying adipogenesis pathways and producing functional adipocytes.     

Altered activity in cultured cells caused by contaminants in tubes widely used for blood collection and serum preparation

Summary Cell culture has been recognized as an extremely sensitive system for measuring the toxicity of various materials. A study was done to determine whether the type of tube used to collect blood or store human serum might affect results in experiments requiring blood drawn into such tubes. In order to test tubes for contaminants that might alter cellular activity, a variety of commercially available tubes used for collection of blood and storage of serum were shaken while containing culture medium with fetal bovine serum. The medium was then applied to 3T3 fibroblasts in culture. Measuring incorporation of tritiated thymidine into DNA in log phase cells as an index of cellular proliferation, it was found that medium containing serum preincubated in tubes routinely used for blood collection could be extremely toxic. The same types of tube were also used to prepare human serum. When serum from some of the tubes was applied to 3T3 fibroblasts, a stimulatory effect was observed, perhaps caused by selective adsorption of inhibitory components of the blood or serum by various tubes. It is, therefore, crucial in a properly controlled experiment using serum in vitro to collect blood in tubes that exert no toxic or stimulatory effects in the assay or, at least, to be consistent in one’s choice of tube. None of the tubes used for storage of serum showed significant effects in our assay.     

Human umbilical cord blood cells improve cardiac function after myocardial infarction

Human umbilical cord blood (UCB) contains an abundance of immature stem/progenitor cells and has been clinically used as an alternative to bone marrow transplantation. In addition, cord blood can be obtained non-invasively, in contrast to invasive bone marrow aspiration. We investigated the potential of human UCB CD34(+) cells to improve cardiac function following myocardial infarction. Myocardial infarction was induced in Wistar rats by ligation of the left coronary artery. Either 2x10(5) human UCB CD34(+) cells or equivalent cell-free medium was injected into the injured myocardium of the rats following induction of myocardial infarction. CD34(+) cell transplantation significantly improved ventricular function as compared to the control group. Immunofluorescence staining for human CD34, CD45, and PECAM-1 revealed surviving cells in the myocardium. Our findings suggest that transplanted human cells survived and improved cardiac function following myocardial infarction. These results may show the usefulness of UCB CD34(+) cells for myocardial infarction.     

Statistical analysis of functional response experiments

The aim of this paper is to highlight the benefits of a full data analysis of a functional response data set, compared to the usual one‐pass regression analysis. In a biological control setting where the choice of organism is often based on comparative studies of the functional responses, it is imperative to have both reliable estimates and a feeling of the degree of confidence one is willing to put on the figures. We analyzed a data set involving the freshwater predator Notonecta glauca (Hemiptera) preying on Asellus aquaticus during 24 h. The specific aim of the analysis was to test whether the functional response is of type II or type III. The different stages of a complete analysis are (1) a preliminary inspection of the data, (2) model building, (3) a model check and (4) a combination of the results with independent information. We argue that the analysis is best done with the predation rate as response and define a test for the location of its maximum. The existence of a maximum is typical for type III functional response. We explain why the binomial distribution is a natural error distribution, and how to implement the regression analysis within the family of generalized linear models using two competing link functions, the logit and the reciprocal. There is marked overdispersion which increases with increasing prey numbers. We use prior weights to take account of it. Using all available data, a type III functional response is warranted with the reciprocal link, but not with the logit link Model checks using Pearson residuals and regression diagnostics based on point deletions show that three points have a particularly strong influence on the parameter estimates. If these are deleted, the functional response type III is then warranted for both link functions. The complete analysis enables us to determine the various degrees of uncertainty and to draw biological conclusions with corresponding confidence. We are convinced that the data set shows a type III functional response, but we are less sure about which link function to choose. Furthermore, the marked overdispersion at high density, the regression diagnostics, as well as independent information on a change in the behaviour of the prey at high density, indicate that the experimental conditions may have changed as a function of the prey density.     

Tissue engineering of blood vessels with endothelial cells differentiated from mouse embryonic stem cells

Endothelial cells (TEC3 cells) derived from mouse embryonic stem (ES) cells were used as seed cells to construct blood vessels. Tissue engineered blood vessels were made by seeding 8 X 106 smooth muscle cells (SMCs) ob-tained from rabbit arteries onto a sheet of nonwoven polyglycolic acid (PGA) fibers, which was used as a biode-gradable polymer scaffold. After being cultured in DMEM medium for 7 days in vitro, SMCs grew well on the PGA fibers, and the cell-PGA sheet was then wrapped around a silicon tube, and implanted subcutaneously into nude mice. After 6～8 weeks, the silicon tube was replaced with another silicon tube in smaller diameter, and then the TEC3 cells (endothelial cells differentiated from mouse ES cells) were injected inside the engineered vessel tube as the test group. In the control group only culture medium was injected. Five days later, the engineered vessels were harvested for gross observation, histological and immunohistochemical analysis. The preliminary results demonstrated that the SMC-PGA construct could form a tubular structure in 6-8 weeks and PGA fibers were completely degraded. Histological and immunohistochemical analysis of the newly formed tissue revealed a typical blood vessel structure, including a lining of endothelial cells (ECs) on the lumimal surface and the presence of SMC and collagen in the wall. No EC lining was found in the tubes of control group. Therefore, the ECs differentiated from mouse ES cells can serve as seed cells for endothelium lining in tissue engineered blood vessels.