A cell number-counting factor regulates group size in Dictyostelium by differentially modulating cAMP-induced cAMP and cGMP pulse sizes

A secreted counting factor (CF), regulates the size of Dictyostelium discoideum fruiting bodies in part by regulating cell-cell adhesion. Aggregation and the expression of adhesion molecules are mediated by relayed pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. A 1-min exposure of cells to purified CF increases the cAMP-induced cAMP pulse. CF does not affect the cAMP receptor or its interaction with its associated G proteins or the translocation of the cytosolic regulator of adenylyl cyclase to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, whereas reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The results suggest that regulating cell-cell adhesion, the cGMP pulse size, or the cAMP pulse size can control group size and that CF regulates all three of these independently.     

Size measurements on isolated rat heart cells using coulter analysis and light scatter flow cytometry

Isolated ventricular muscle cells from the adult rat heart have been examined by both Coulter analysis and light scatter flow cytometry. The dispersed cell preparations contain two main cell types: viable, rod-shaped cells and damaged, round cells. Coulter analytical techniques provided statistical data on cell volume for both cell types. The contribution of each population to the Coulter pulse height distributions were separated by a subtraction method using data obtained from digitonin-treated preparations that contain only round cells. A shape factor for cells aligned with the flow direction was computed from light microscope measurements and the effects of cell orientation within the Coulter aperture were approximately assessed. The estimated volumes for intact myocytes compare favourably with those reported in the literature. No significant size difference was observed between fresh and fixed cells.Narrow angle, forward light scatter measurements were made on individual cells flowing across a focused laser beam. Both scatter pulse height and pulse width (pulse duration) distributions were collected. Values for myocyte length calculated from pulse width information agree well with published data and confirm that the hydrodynamic forces in the flow system produced alignment of the cells with the flow direction. Scatter pulse width distributions reveal two distinct peaks assignable to either rod or round cells. Preliminary electronic gating experiments, using pulse height signals, suggest that signals derived from round cells could be eliminated entirely using a gating regime based on pulse width. This would enable flow cytometric measurements to be made on only the intact myocytes present in heterogeneous preparations.     

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.     

Size of the pores created by an electric pulse: Microsecond vs millisecond pulses

Here, the sizes of the pores created by square-wave electric pulses with the duration of 100μs and 2ms are compared for pulses with the amplitudes close to the threshold of electroporation. Experiments were carried out with three types of cells: mouse hepatoma MH-22A cells, Chinese hamster ovary (CHO) cells, and human erythrocytes. In the case of a short pulse (square-wave with the duration of 100μs or exponential with the time constant of 22μs), in the large portion (30-60%) of electroporated (permeable to potassium ions) cells, an electric pulse created only the pores, which were smaller than the molecule of bleomycin (molecular mass of 1450Da, r≈0.8nm) or sucrose (molecular mass of 342.3Da, radius-0.44-0.52nm). In the case of a long 2-ms duration pulse, in almost all cells, which were electroporated, there were the pores larger than the molecules of bleomycin and/or sucrose. Kinetics of pore resealing depended on the pulse duration and was faster after the shorter pulse. After a short 100-μs duration pulse, the disappearance of the pores permeable to bleomycin was completed after 6-7min at 24-26°C, while after a long 2-ms duration pulse, this process was slower and lasted 15-20min. Thus, it can be concluded that a short 100-μs duration pulse created smaller pores than the longer 2-ms duration pulse. This could be attributed to the time inadequacy for pores to grow and expand during the pulse, in the case of short pulses.     

Ability of repetitive Ca2+ spikes to stimulate prolactin release is frequency dependent

Measurements of concentrations of cytosolic free Ca2+ ([Ca2+]c) in individual cells has frequently demonstrated periodic transients in [Ca2+]c rather than sustained elevated levels. To determine in anterior pituitary cells if such short and repetitive [Ca2+]c transients stimulated prolactin release, we used a perifusion system with cells loaded with the fluorescent Ca2+-indicator, indo-1. A one second pulse of 100 mM KCl caused an increase in [Ca2+]c with a half peak width of about 18 seconds and an almost coincident increase in prolactin secretion. Subsequent pulses of KCl each caused increases in [Ca2+]c and prolactin release that were the same as the first, up to a pulse frequency of one every two minutes. Increasing the frequency to 1 pulse every minute or 1 pulse every 30 seconds, however, resulted in a serial decline in the amount of prolactin released by each pulse even though each pulse caused a similar peak Ca2+ response. These findings demonstrate that cells become adapted to transient increases in [Ca2+]c of the same magnitude so that they no longer release prolactin if the increases in [Ca2+]c occur frequently enough. Cells may use frequency-encoded Ca2+ signals to stimulate release of prolactin at low frequency to prevent the adaptation that occurs at higher frequencies.     

Mechanism of T cell activation. II. Antigen- and lectin-dependent acquisition of responsiveness to TCGF is a nonmitogenic, active response of resting T cells

Small resting T cells, which do not respond to T cell growth factor (TCGF), acquire responsiveness upon a short (4-hr) pulse of specific ligands by presenting growth receptors for TCGF. The results demonstrate that the same mechanisms operate in the specific induction of primary MLR in that a 5-hr MLR is sufficient to render the responder cells reactive to TCGF. Furthermore, the results demonstrate that an active "response" by the resting T cells is required for expression of functional growth receptors, as demonstrated by the fact that: 1) a 4-hr pulse of concanavalin A (Con A) at 4 degrees C did not result in gain of reactivity to TCGF, whereas a 4-hr pulse at 37 degrees C did; 2) this metabolic requirement for acquisition of responsiveness to TCGF was not due to a secondary requirement for cap-formation of Con A-binding membrane structures, as normal responses were observed in the presence of cytochalasin B (cyt B); 3) the process of Con A-induced acquisition of susceptibility to TCGF was puromycin sensitive.     

Two components of a secreted cell number-counting factor bind to cells and have opposing effects on cAMP signal transduction in Dictyostelium

A secreted 450-kDa complex of proteins called counting factor (CF) is part of a negative feedback loop that regulates the size of the groups formed by developing Dictyostelium cells. Two components of CF are countin and CF50. Both recombinant countin and recombinant CF50 decrease group size in Dictyostelium. countin- cells have a decreased cAMP-stimulated cAMP pulse, whereas recombinant countin potentiates the cAMP pulse. We find that CF50 cells have an increased cAMP pulse, whereas recombinant CF50 decreases the cAMP pulse, suggesting that countin and CF50 have opposite effects on cAMP signal transduction. In addition, countin and CF50 have opposite effects on cAMP-stimulated Erk2 activation. However, like recombinant countin, recombinant CF50 increases cell motility. We previously found that cells bind recombinant countin with a Hill coefficient of approximately 2, a KH of 60 pm, and approximately 53 sites/cell. We find here that cells also bind 125I-recombinant CF50, with a Hill coefficient of approximately 2, a KH of approximately 15 ng/ml (490 pm), and approximately 56 sites/cell. Countin and CF50 require each other's presence to affect group size, but the presence of countin is not necessary for CF50 to bind to cells, and CF50 is not necessary for countin to bind to cells. Our working hypothesis is that a signal transduction pathway activated by countin binding to cells modulates a signal transduction pathway activated by CF50 binding to cells and vice versa and that these two pathways can be distinguished by their effects on cAMP signal transduction.     

低压脉冲电泳介导的外源基因转化部分酶解的水稻小细胞团

开发了一类新型的低压脉冲电泳法介导外源基因进入水稻细胞的转化系统。本系统以水稻部分酶解小细胞团为受体,采用低压脉冲电泳推动质核DNA进入水稻细胞。以报告基因GUS酶活性为指标,借以测定转化了的水稻细胞。最佳的组合处理可以获得8.2％的转化频率。文中对低压脉冲电泳转移外源基因的条件亦作了讨论。     

RNA synthesis in whole cells and protoplasts of centaurea: a comparison

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [³H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.     

Reversible binding of phytomitogens to lymphocytes

The reversible binding of phytohaemagglutinin and concanavalin A to lymphoyctes and red cells was studied by radiolabelling techniques. The quantity of mitogen bound was dependent on the temperature, concentration and duration of the mitogen pulse. Dissociation of mitogen occurred rapidly from red cells and lymphocytes and the kinetics of release from lymphoid cells displayed two components with different half lives. There was suggestive evidence that the binding component with the longer half life was related to lymphocyte activation. The induction of DNA synthesis in lymphocytes required at least 20-hr exposure to the mitogens. These data indicate that the binding of mitogen is a complex reversible process in which association and dissociation are critically dependent on the temperature, concentration and duration of the mitogen pulse.     

Activation of lymphocytes by concanavalin A requires calcium ions.

Mouse spleen cells were exposed to a short pulse of the mitogenic lectin concanavalin A (con A). After removal of con A mitogenesis was measured by the incorporation of tritiated thymidine into DNA. It was found: (a) the number of cells responding to con A was proportional to the time of exposure to con A; (b) exposure of cells to con A in the absence of extracellular calcium failed to initiate mitogenesis; (c) for a mitogenic effect an extracellular calcium concentration greater than 10(-5)M was required during the time that the cells were exposed to con A.     

Performance analysis of a dual-buffer architecture for digital flow cytometry.

BACKGROUND: Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis. METHODS: The second-generation system incorporates fast digitization of analog pulse waveforms, instantaneous pulse detection hardware, and a novel dual-buffering scheme. A mathematical model of the system was developed to theoretically compute the capture-rate performance. RESULTS: The capture rate of the system was theoretically analyzed and empirically measured. Under typical conditions, a capture rate of 8,000 pulses/s was experimentally achieved. CONCLUSIONS: Based on these results, the dual-buffer architecture shows great potential for use in flow cytometry.     

Kinetics of ultrastructural changes during electrically induced fusion of human erythrocytes

Summary The sequence of events during the electrically induced fusion of human erythrocytes was studied by rapid quench freeze-fracture electron microscopy. A single electric field pulse was used to induce fusion of human erythrocytes treated with pronase and closely positioned by dielectrophoresis. The electronic circuit was coupled to a rapid freezing mechanism so that ultrastructural changes of the membrane could be preserved at given time points. Pronase treatment enabled adjacent cells to approach each other within 15 nm during dielectrophoresis. The pulse caused a brief disruption of the aqueous boundaries which separated the cells. Within 100 msec following pulse application, the fracture faces exhibited discontinuous areas which were predominantly free of intramembranous particles. At 2 sec after the pulse, transient point defects attributed to intercellular contact appeared in the same membrane areas and replaced the discontinuous areas as the predominant membrane perturbation. At 10 sec after the pulse, the majority of the discontinuous areas and point defects disappeared as the intercellular distance returned to approximately 15 to 25 nm, except at sites of cytoplasmic bridge formation. Intramembranous particle clearing was observed at 60 sec following pulse application in discrete zones of membrane fusion.     

Early postnatal proliferation of the retinal pigment epithelium cells in the mouse

A burst of proliferative activity with a maximum of DNA-synthesizing cells on the first day after birth was found in the central zone of the retinal pigment epithelium (RPE) in albino mice from the moment of birth to 9 days of life using radioautography with 3H-thymidine pulse labelling. During this period the central RPE zone, which consists in newborns of mononuclear cells by 95%, gradually transforms in a population with predominance of binuclear cells and fluctuations in the index of labelled nuclei (after the kinetics of cell population in the central RPE zone is similar in mice and rats both in accumulation of binuclear cells and fluctuations in the index of labelled nuclei (after pulse labelling), except that in mice the peak of the index of labelled nuclei is observed earlier than in rats.     

Combined Pulse Electroporation – A Novel Strategy for Highly Efficient Transfection of Human and Mouse Cells

The type of a nucleic acid and the type of the cell to be transfected generally affect the efficiency of electroporation, the versatile method of choice for gene regulation studies or for recombinant protein expression. We here present a combined square pulse electroporation strategy to reproducibly and efficiently transfect eukaryotic cells. Cells suspended in a universal buffer system received an initial high voltage pulse that was continuously combined with a subsequent low voltage pulse with independently defined electric parameters of the effective field and the duration of each pulse. At comparable viable cell recoveries and transfection efficiencies of up to 95% of all cells, a wide variety of cells especially profited from this combined pulse strategy by high protein expression levels of individual cells after transfection. Long-term silencing of gene expression by transfected small interfering RNA was most likely due to the uptake of large nucleic acid amounts as shown by direct detection of fluorochromated small interfering RNA. The highly efficient combined pulse electroporation strategy enables for external regulation of the number of naked nucleic acid molecules taken up and can be easily adapted for cells considered difficult to transfect.     

Electroporation of extraneous proteins into CHO cells: Increased efficacy by utilizing centrifugal force and microsecond electrical pulses

A novel electroporation system employing an oscillating electric pulse and centrifugal force was used to introduce extraneous proteins into CHO cells. Following the electrical pulse, the compression and subsequent rebound induced by the centrifugal acceleration and deceleration, respectively, enhanced protein uptake, presumably by a hydrodynamic pumping of extracellular solutions through the permeabilized membrane. Protein uptake was quantitated by measuring the amount of radiolabeled, extraneous, CHO proteins introduced into unlabeled CHO cells. The amount of protein introduced into electroporated CHO cells was enhanced up to four-fold by a combination of electric pulse and centrifugal force compared to that introduced by electric pulse only. The optimum gradient of centrifugal force (GCF, temporal change of centrifugal force) was 590 and -470 g/s during acceleration and deceleration, respectively. The optimum electric field was 5 kV/cm with a 30-microsecond pulse length. At this optimum electroporation condition, approximately 5 pg of proteins (up to 200 kDa molecular weight) were introduced per CHO cell. These same settings also permitted electroporation of other membrane impermeable substances including propidium iodide and ethidium bromide. Introduction of extraneous materials into the cytoplasm during electroporation was confirmed by the ability of anti alpha-tubulin to stain the microtubules and propidium iodide and ethidium bromide to stain the nuclei. Cells electroporated with optimum device settings exhibited no significant decrease in clonogenic survival.     

Photoresponses of Halobacterium salinarum to repetitive pulse stimuli.

Halobacterium salinarum cells from 3-day-old cultures have been stimulated with different patterns of repetitive pulse stimuli. A short train of 0.6-s orange light pulses with a 4-s period resulted in reversal peaks of increasing intensity. The reverse occurred when blue light pulses were delivered as a finite train: with a 3-s period, the response declined in sequence from the first to the last pulse. To evaluate the response of the system under steady-state conditions of stimulation, continuous trains of pulses were also applied; whereas blue light always produced a sharply peaked response immediately after each pulse, orange pulses resulted in a declining peak of reversals that lasted until the subsequent pulse. An attempt to account for these results in terms of current excitation/adaptation models shows that additional mechanisms appear to be at work in this transduction chain.     

Evaluation of intensity and pulse width of different moderators for designing a new diffractometer for protein crystals with large unit cells in J-PARC/MLF

We plan to design a high-resolution biomacromolecule neutron time-of-flight diffractometer, which allows us to collect data from crystals with unit cells above 250 Å, in the materials and life science experimental facility at the Japan Proton Accelerator Research Complex. This new diffractometer can be used for a detailed analysis of large proteins such as membrane proteins and supermolecular complex. A quantitative comparison of the intensity and pulse width of a decoupled moderator (DM) against a coupled moderator (CM) considering the pulse width time resolution indicated that the DM satisfies the criteria for our diffractometer rather than the CM. The results suggested that a characteristic feature of the DM, i.e., narrow pulse width with a short tail, is crucial for the separation of Bragg reflections from crystals with large unit cells. On the other hand, it should be noted that the weak signals from the DM are buried under the high-level background caused by the incoherent scattering of hydrogen atoms, especially, in the case of large unit cells. We propose a profile-fitting integration method combined with the energy loss functions and a background subtraction method achieved by employing the statistics-sensitive nonlinear iterative peak-clipping algorithm.     

Specificity of the lethal and mutagenic actions of pico-second laser pulses of 532-nm wavelength

A study was made of the lethal effect of pulse laser (second harmonic Nd+3:YAG laser of 532 nm, pulse length 3.3.10(-11) s, peak intensity from 4.10(12) to 1.10(14) W/m2) on HeLa cells at the phases of active and stationary growth, and lethal and mutagenic effects of this radiation on E. coli cells. As was shown, HeLa cells at both growth phases and E. coli cells exhibited low sensitivity to laser radiation at lambda = 532 nm.