Kinetic properties of DM-nitrophen binding to calcium and magnesium

Caged-Ca(2+) compounds such as nitrophenyl-EGTA (NP-EGTA) and DM-nitrophen (DMn) are extremely useful in biological research, but their use in live cells is hampered by cytoplasmic [Mg(2+)]. We determined the properties of Ca(2+) release from NP-EGTA and DMn by using Oregon green BAPTA-5N to measure changes in [Ca(2+)] after ultraviolet flash photolysis in vitro, with or without Mg(2+) present. A large fraction (65%) of NP-EGTA, which has a negligible Mg(2+) affinity, uncages with a time constant of 10.3 ms, resulting in relatively slow increases in [Ca(2+)]. Uncaging of DMn is considerably faster, but DMn has a significant affinity for Mg(2+) to complicate the uncaging process. With experimentally determined values for the Ca(2+) and Mg(2+) binding/unbinding rates of DMn and NP-EGTA, we built a mathematical model to assess the utility of NP-EGTA and DMn in rapid Ca(2+)-uncaging experiments in the presence of Mg(2+). We discuss the advantages and disadvantages of using each compound under different conditions. To determine the kinetics of Ca(2+) binding to biologically relevant Ca(2+) buffers, such as Ca(2+)-binding proteins, the use of DMn is preferable even in the presence of Mg(2+).     

Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen.

Nitrophenyl-EGTA and DM-nitrophen are Ca2+ cages that release Ca2+ when cleaved upon illumination with near-ultraviolet light. Laser photolysis of nitrophenyl-EGTA produced transient intermediates that decayed biexponentially with rates of 500,000 s-1 and 100,000 s-1 in the presence of saturating Ca2+ and 290,000 s-1 and 68,000 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. Laser photolysis of nitrophenyl-EGTA in the presence of Ca2+ and the Ca2+ indicator Ca-orange-5N produced a monotonic increase in the indicator fluorescence, which had a rate of 68,000 s-1 at pH 7.2 and 25 degrees C. Irradiation of DM-nitrophen produced similar results with somewhat slower kinetics. The transient intermediates decayed with rates of 80,000 s-1 and 11,000 s-1 in the presence of Ca2+ and 59,000 s-1 and 3,600 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. The rate of increase in Ca(2+)-indicator fluorescence produced upon photolysis of the DM-nitrophen: Ca2+ complex was 38,000 s-1 at pH 7.2 and 25 degrees C. In contrast, pulses in Ca2+ concentration were generated when the chelator concentrations were more than the total Ca2+ concentration. Photoreleased Ca2+ concentration stabilized under these circumstances to a steady state within 1-2 ms.     

Calcium regulation by and buffer capacity of molluscan neurons during calcium transients

The properties of Ca-regulation and -buffering of physiological levels of Ca-transients were examined in the soma of Archidoris monteryensis neurons. The rate of recovery from a Ca-transient was examined with two experimental protocols; in one the pulse duration was kept constant and its amplitude was varied, and in the other the duration was varied while the amplitude was kept constant. These experiments revealed that the recovery from a Ca-transient was approximately a first order process and the apparent first order rate constant was dependent on the duration of Ca-influx. The calcium buffer capacity of the cytoplasm was determined by an indirect method which utilised measured amounts of intracellular EGTA to reduce transient changes in free calcium. An equation for the cytoplasmic buffer capacity was derived on the assumption that the capacities of exogenous and endogenous Ca buffers summate linearly. The resting cytoplasmic Ca buffer capacity was 45.2 microM/delta pCa, when it was assumed that the incoming Ca diffuses a distance of 10 microns into the cytoplasm. For a diffusion distance of 5 microns it was 34.5 microM/delta pCa. In both cases, the buffer capacity increased with an increase in the size of Ca transient.     

SH—SY5Y细胞的钙缓冲研究

目的：研究SH－SY5Y神经杂交瘤细胞的钙缓冲能力。方法：通过膜片钳手段,测量未分化的SH－SY5Y细胞钙离子通道电流;并应用显微荧光测量游离钙离子浓度和高钾去极化的方法,研究胞内Ca^2 浓度上升后浓度恢复的动力学过程。结果：未分化的SH－SY5Y细胞存在钙离子通道电流,在刺激时间间隔较短时（＜150s）,胞内钙浓度的恢复过程会由于缓冲机制的饱和而变慢;而时间间隔＞150s时,缓冲物质则可以基本恢复使得胞内钙的恢复过程基本保持不变。结论：钙缓冲蛋白在细胞内钙浓度的调节中起重要作用。     

Magnesium binding to DM-nitrophen and its effect on the photorelease of calcium

The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.     

High speed two-photon imaging of calcium dynamics in dendritic spines: consequences for spine calcium kinetics and buffer capacity

Rapid calcium concentration changes in postsynaptic structures are crucial for synaptic plasticity. Thus far, the determinants of postsynaptic calcium dynamics have been studied predominantly based on the decay kinetics of calcium transients. Calcium rise times in spines in response to single action potentials (AP) are almost never measured due to technical limitations, but they could be crucial for synaptic plasticity. With high-speed, precisely-targeted, two-photon point imaging we measured both calcium rise and decay kinetics in spines and secondary dendrites in neocortical pyramidal neurons. We found that both rise and decay kinetics of changes in calcium-indicator fluorescence are about twice as fast in spines. During AP trains, spine calcium changes follow each AP, but not in dendrites. Apart from the higher surface-to-volume ratio (SVR), we observed that neocortical dendritic spines have a markedly smaller endogenous buffer capacity with respect to their parental dendrites. Calcium influx time course and calcium extrusion rate were both in the same range for spines and dendrites when fitted with a dynamic multi-compartment model that included calcium binding kinetics and diffusion. In a subsequent analysis we used this model to investigate which parameters are critical determinants in spine calcium dynamics. The model confirmed the experimental findings: a higher SVR is not sufficient by itself to explain the faster rise time kinetics in spines, but only when paired with a lower buffer capacity in spines. Simulations at zero calcium-dye conditions show that calmodulin is more efficiently activated in spines, which indicates that spine morphology and buffering conditions in neocortical spines favor synaptic plasticity.     

Quality control of an unreliable flexible manufacturing system with scrapping and infinite buffer capacity

Manufacturing multiple part types on a flexible manufacturing system (FMS) is increasingly becoming a rule rather than an exception. In such systems, attention has been drawn to the application of zero-defect technologies. However, in practice, this goal has remained elusive and costly. As a result, even though FMSs may be more reliable, producing fewer defective parts, system complexity and more stringent quality standards are rendering quality control in FMSs potentially useful. The goals of this article are threefold. First, we introduce a procedure for measuring and managing the in-process quality control of an FMS, which is described by an Open Queueing Network (OQN), bridging thereby a gap between queueing theory and quality control. Second, by focusing attention on the potential unreliabilities of FMSs, we provide some managerial insights regarding the role, position, and distribution of the quality control effort in an FMS. Finally, we stress the intricate relations between an FMS's operating characteristics and the manufactured quality and its control. Using numerical analyses, we draw some inferences regarding the design of such FMSs when both quality and quantity issues in the FMSs are considered. These simultaneous considerations of quantity and quality flows in an FMS have not been previously considered in the study of FMSs.     

Cytoplasmic 5'-nucleotidase catalyzes acyclovir phosphorylation

A cytoplasmic 5'-nucleotidase (EC 3.1.3.5) can catalyze the phosphorylation of inosine (Worku, Y., and Newby, A.C. (1982) Biochem. J. 205, 503-510). This enzyme was purified to determine whether it could catalyze the formation of trace levels of phosphorylated acyclovir (ACV), a nucleoside analog with antiherpes activity. Acyclovir phosphorylating activity from rat liver co-chromatographed with the enzyme throughout the 1200-fold purification and through size exclusion chromatography or polyacrylamide-gel electrophoresis. In addition, the pH optimum, ATP stimulation, and phosphate inhibition of the ACV phosphorylating activity paralleled those of the 5'-nucleotidase. Finally, ACV phosphorylation was competitively inhibited by inosine (Kis = 6.5 mM; K'm (inosine) = 5.0 mM). This was consistent with phosphorylation at a common catalytic site. In addition to inosine and ACV, the guanine derivatives Guo, dGuo, 9-beta-D-arabinofuranosylguanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine were substrates for the enzyme. The relative phosphorylation rates were, respectively, 100, 0.7, 19, 4, 0.3, and 0.7, at 0.1 mM phosphate acceptor. Approximate K'm values were, respectively, 5, 90, 10, 10, greater than 100, and greater than 100 mM. Although the substrate activity of ACV with the 5'-nucleotidase was inefficient, it appeared to be sufficient to account for the small amounts of ACV phosphates formed in uninfected cells.     

Conductivity and buffer capacity of potato juice as correlated with potassium supply to the plant

Summary Electrolytic conductivity and buffer capacity of the press juice of the potato tuber are almost exclusively determined by the potassium supply to the plant and by the form in which the potassium fertilizer is given (especially in respect of the chlorine uptake of the plant).     

Cytoplasmic calcium stimulates exocytosis in a plant secretory cell

Although exocytosis is likely to occur in plant cells, the control of this process is the subject of speculation, as no direct measurements of vesicle fusion to the plasma membrane have been made. We used the patch clamp technique to monitor the secretory activity of single aleurone protoplasts by measuring membrane capacitance (C_m), while dialyzing the cytosol with different Ca²⁺ containing solutions. Secretory activity increased with [Ca²⁺]_i ~ 1 μM. This demonstrates directly the existence of exocytosis in plant cells, and suggests that both plant and animal cells share common mechanisms (cytosolic Ca²⁺) for the control of exocytotic secretion.     

Structural transitions in polycytidylic acid: proton buffer capacity data

The pH-dependences of proton buffer capacity of poly(C) were computed on the basis of the literature data. In these curves there were observed four peaks: two narrow and two wide ones. The first narrow peak reflects the process of cooperative formation of double helices, which is induced by protonation of the N3 atom of nucleotide bases. The first wide peak is assigned to noncooperative process of poly(C) double helices protonation at the N3 nitrogen atom. It is proposed that the second wide peak corresponds to noncooperative protonation of the neutral cytosine bases at the oxygen atom. This reaction causes cooperative dissociation of the poly(C) double helices. The second narrow peak reflects the dissociation process.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.     

Cytoplasmic manifestation of the nuclear membrane's hormone binding capacity during cell division

Summary Binding of insulin and thyrotropic hormone (TSH) to the nuclear membrane of Chang liver cells was demonstrated by qualitative and quantitative cytofluorimetry, which failed to substantiate a similar binding affinity for BSA. It appears that in the dividing cell the binding structures (receptors) of the nuclear membrane migrate in the cytoplasm together with the chromosomes by the end of the prophase and become reorganized in the nucleus around the telophase. The fluorescence which indicated binding also appeared in the midbody region during division of the two daughter cells. These experimental observations strongly suggest that, after cell division, only part of the nuclear membrane's receptor complement has to be resynthesized in the daughter cells, because the receptor number required by a single cell is conserved in cytoplasmic membrane details of nuclear membrane origin.