Measuring Absorbance Changes Research Articles

High Performance Detection of Alzheimer’s Disease Biomarkers Based on Localized Surface Plasmon Resonance

In this study, we fabricated sensors to detect Alzheimer’s disease (AD) biomarkers – amyloid beta (1-42) (Aβ1-42) based on localized surface plasmon resonance (LSPR). The sensors were consisted of ligand-exchanged gold nanoparticles (Au NPs) deposited on polyethylene terephthalate substrate using Langmuir-Blodgett (LB) technique. Monoclonal antibodies (anti-Aβ1-42) were then immobilized as a conjugate form with biotin onto the LB films of ligand-exchanged Au NPs by the aid of streptavidin. The attachment of the biomarkers to the antibodies immobilized on the LB films was detected by measuring absorbance change of plasmonic response peak. The sensor structure was optimized by comparing the results for the Au NPs LB films with different size and film thickness. The optimized sensor was used to detect biomarker at different concentrations in the buffer solution and a diluted cerebrospinal fluid (CSF) solution. As results, the sensor was able to detect even a trace amount of Aβ1-42 as small as 1 pg/ml from the CSF. This prototype sensor has a great potential because it shows several advantages of facile and inexpensive fabrication, and early detection of the AD.

Pharmacodynamic testing and new validated HPLC method to assess the interchangeability between multi-source orlistat capsules.

BackgroundOrlistat is an irreversible inhibitor of the lipase enzyme that prevents trigylcerides from being digested, thereby inhibiting triglyceride hydrolysis and absorption. The resultant reduced calorie uptake enables a positive effect on weight control. Systemic absorption of the drug is, therefore, not necessary for its mode of action. An alternative in vitro study (pharmacodynamic) has been introduced for this drug, as in vivo bioavailability studies are irrelevant with regard to the achievement of the product’s intended purposes.ObjectivesTo develop a new validated high-performance liquid chromatography (HPLC) method for the analysis of orlistat and to assess the potency and equivalence of three orlistat formulations using the pharmacodynamic method as a surrogate indicator of pharmaceutical interchangeability.MethodsA new HPLC method was developed for the analysis and for the dissolution studies of orlistat in capsules. Pancreatic lipase activity was measured for three different capsule products: Orlislim®, Slimcare®, and Xenical®, G1, G2, and the brand, respectively. Porcine pancreatic lipase and p-nitrophenyl butyrate (PNPB) were placed in a pH 7.4 reaction buffer at 37°C, and substrate hydrolysis was monitored by measuring absorbance changes at 410 nm; this was repeated on six capsules of each product. The inhibition was expressed by the concentration of product, which inhibited 50% of the activity of pancreatic lipase (IC50).ResultsThe new analytical method was suitable for orlistat analysis. Values of IC50 from regression lines and equations were 6.14, 8.43, and 7.80 μg/mL for Orlislim®, Xenical®, and Slimcare®, respectively.ConclusionPharmacodynamic studies of lipase inhibition could be used to support in vitro dissolution, which demonstrates interchangeability between generic and branded orlistat capsules. Moreover, it could be suggested as an alternative tool to bioequivalence studies for orlistat oral products.

Investigating changes in the redox state of Photosystem I at low pH

Changes in the redox state of Photosystem I (PSI) were studied in spinach leaf discs suspended in buffers of different pH (pH 7.5, 6.5, 5.5 and 4.5). By measuring absorbance changes at 820nm, it was observed that under normal conditions, the electrons were supplied by Photosystem II (PSII) for the photo-oxidation of P700 while in the presence of DCMU when electrons coming from PSII are blocked, cyclic electron flow (CEF) around PSI was the major source for the absorbance changes observed at 820nm. This was supported by complete inhibition in the reduction of both single turnover (ST) area and multiple turnover (MT) area, in the presence of DCMU, which is generally filled up by the electrons coming from PSII. In the absence of DCMU, the intersystem electron pool or plastoquinone (PQ) pool was increased at low pH which was probably due to enhanced cyclic electron flow around PSI. Our results also suggest that at low pH, in the absence of DCMU, the major contribution for faster dark re-reduction of P700+ is attributed mainly by PSII and CEF PSI while in the presence of DCMU, the significant contribution is provided by CEF PSI and other stromal components.

Micellar sensitized catalytic kinetic spectrophotometry for highly accurate and reproducible determination of V(IV) and V(V)

A highly accurate and reproducible micellar sensitized kinetic method was proposed for determination of V(VI). The method is based on its catalytic effect on the oxidation of Coomassie brilliant blue R 250 (CBB+) by bromate at pH 2.0. The reaction was monitored spectrophotometrically by measuring absorbance change with a fixed-time method of 5 min at 594 and 552 nm with and without surfactant. The variables influencing the calibration sensitivity were extensively investigated, and the optimal conditions were established. The linear calibration range was 10–1,600 μg·L−1 with a relative SD ranging from 0.35 % to 3.35 % (for five replicate measurements of 75, 500, 1,000, and 1,500 μg·L−1) and a detection limit of 3.8 μg·L−1. The selectivity was also investigated, and greatly enhanced by suitable masking agents. The method was successfully applied to the analysis of V(IV) in presence of excess V(V) up to 25 fold in environmental waters with the recoveries of 100.0 %–102.8 % for V(IV) and 95.7 %–99.7 % for total V. Its accuracy was validated by analysis of certified reference material via the present kinetic method and standard flame atomic absorption spectrometric method after extractive preconcentration with good agreement between certified and found values.

A novel microwell-based analytical technique for studying ferrous iron biooxidation activity

The broad range of complexities in bioleaching includes the use of mixed microbial communities with diversity of species and strains with different windows of operating conditions. Empirical approaches to characterise these currently use cumbersome experimental systems; hence the need to develop a high throughput research tool, analogous to the techniques used in high throughput pharmaceutical research. In this study, a microwell research tool was evaluated as a growth and measurement tool for mixed autotrophic bioleaching cultures. The tool was assessed by comparing its performance to conventional shake flask apparatus. Mixed mesophilic cultures of predominantly Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans were used. Growth and ferrous iron oxidation kinetics were quantified and assessed. Microwell plates performed similarly to conventional shake flasks with respect to growth and iron oxidation kinetics. The microwell plate apparatus was also used as a measurement system in combination with a microwell plate reader (measuring absorbance change at 428nm over time). Progressive colour change of growth experiments correlated to ferrous iron oxidation within a defined operating window. We conclude that, using this measurement as a proxy for trends iron oxidation, the microwell research tool is well-suited for high throughput scoping studies to map operating windows for different cultures, in both an unadapted and adapted context. This was confirmed through an activity test utilising fluoride as an inhibitor. Where absorbance measurements at 428nm are used to track oxidation progress, the research tool has limitations with respect to pH (<2.0) and total iron concentration (<8.0gl−1).

Analysis for measuring peroxidase activity in wheat seeds

Peroxidase (POD) activity in wheat seeds using guaiacol-H2O2 as substrate was determined by measuring absorbance changes at 470 nm using a spectrophotometer. The POD activity of wheat was studied under different extraction conditions (three kinds of buffers, three kinds of extraction methods, four kinds of extraction times, six kinds of extraction temperatures and four kinds of extraction eccentricities). Results showed that POD activity using acetate buffer was higher than those of KH2PO4 and sodium phosphate. The assay was repeated three times every 6 h for a single extraction, and the average CVs of the POD values for the buffers were of the order: phosphate buffer (CV=0.031) > sodium acetate (CV=0.0268) > KH2PO4 (CV=0.0168). The repeatability of the extraction method that involved grinding in buffer was lower than those that involved soaking and shaking. The optimum extraction temperature and extraction time were dependent on the cultivars and buffers. No significant differences in the activity of the POD were observed among the different eccentricities of 20000 × g, 15000 × g, 10000 × g and 4000 × g.

Oxidative Cleavage-Based Near-Infrared Fluorescent Probe for Hypochlorous Acid Detection and Myeloperoxidase Activity Evaluation

A near-infrared (NIR) fluorescent probe was synthesized and demonstrated to be highly selective in reaction with hypochlorous acid (HOCl), an endogenous reactive oxygen species (ROS) produced by myeloperoxidase in neutrophils. The reaction with HOCl resulted in the NIR fluorescence quenching at 774 nm and the absorbance decreasing at 710 nm, accompanied by the appearance of a new absorption band at 520 nm. The reaction mechanism was carefully examined and proposed to proceed by initial formation of chlorohydrins and subsequent degradation. This NIR fluorescent probe was successfully applied as a selective and sensitive indicator for HOCl on the basis of either colorimetry or fluorometry, which showed detection limits of 0.13 and 0.70 μM, respectively. In addition, the molecular probe was further demonstrated for NIR fluorescence imaging of HOCl in cells and for evaluating the enzymatic activity of myeloperoxidase in the HOCl generation by measuring absorbance change.

Speciative determination of total V and dissolved inorganic vanadium species in environmental waters by catalytic–kinetic spectrophotometric method

A kinetic determination of V(V) as a catalyst was spectrophotometrically performed by using the indicator reaction of Gallamine blue (GB+) and bromate at pH 2.0. The reaction was followed by measuring absorbance change for a fixed-time of 3min at 537nm. The variables such as reagent concentration, pH, buffer concentration, ionic strength and temperature were optimized to improve the selectivity and sensitivity. Under the optimized conditions, the determination of V(V) was performed in the range 1–100μgL−1 with limits of detection and quantification of 0.31 and 0.94μgL−1. The developed kinetic method is sufficiently sensitive, selective and simple. It was successfully applied to the speciative determination of total V and inorganic dissolved vanadium species, V(V) and V(IV) in environmental water samples. The oxidizing property of permanganate is used to differentiate between V(IV) and V(V) species. The V(IV) content was found by subtracting the V(V) content from those of total V. The recovery is above 95% for V(V) spiked samples. Additionally, the accuracy was validated by analysis of a certified water sample, CRM TMDA-53.3, and the results were in good agreement with the certified value.

Determination of Malic Acid Using a Malate Dehydrogenase Reactor After Purification and Immobilization in Non-Denaturing Conditions and Staining with Ponceau S

Mouse liver cytosolic malate dehydrogenase was separated by non-denaturing two-dimensional electrophoresis and identified. Furthermore, the activity of the enzyme was preserved even after separation, electroblotting onto a membrane and staining with Ponceau S in acidic buffer solution (pH 5.1). Using the membrane-immobilized enzyme, the malic acid content was estimated by measuring absorbance changes due to the conversion of nicotinamide adenine dinucleotide (NAD) to NADH. These results indicate that enzyme reactors can be systematically produced after purification, immobilization and staining with Ponceau S.

Equilibration kinetics in isolated and membrane-bound photosynthetic reaction centers upon illumination: a method to determine the photoexcitation rate

Kinetics of electron transfer, following variation of actinic light intensity, for photosynthetic reaction centers (RCs) of purple bacteria (isolated and membrane-bound) were analyzed by measuring absorbance changes in the primary photoelectron donor absorption band at 865 nm. The bleaching of the primary photoelectron donor absorption band in RCs, following a sudden increase of illumination from the dark to an actinic light intensity of I exp, obeys a simple exponential law with the rate constant (alpha I_{exp } ; + ;k_{text{rec}} ) , in which α is a parameter relating the light intensity, measured in mW/cm2, to a corresponding theoretical rate in units of reciprocal seconds, and k rec is the effective rate constant of the charge recombination in the photosynthetic RCs. In this work, a method for determining the α parameter value is developed and experimentally verified for isolated and membrane-bound RCs, allowing for rigorous modeling of RC macromolecule dynamics under varied photoexcitation conditions. Such modeling is necessary for RCs due to alterations of the forward photoexcitation rates and relaxation rates caused by illumination history and intramolecular structural dynamics effects. It is demonstrated that the classical Bouguer–Lambert–Beer formalism can be applied for the samples with relatively low scattering, which is not necessarily the case with strongly scattering media or high light intensity excitation.

Expression and Characterization of the TNT Nitroreductase of Pseudomonas sp. HK-6 in Escherichia coli

Pseudomonas sp. HK-6 is able to utilize 2,4,6-trinitrotoluene (TNT) as a sole nitrogen source. The pnrB gene of the HK-6 strain was cloned using degenerate primers synthesized on the basis of the sequence information of the terminal amino acids of a previously purified native TNT nitroreductase. The nucleotide sequence of pnrB was 654 bp long, and its deduced polypeptide sequence was composed of 217 amino acid residues with a predicted molecular mass of 24 kDa. To facilitate the purification and characterization of this enzyme, an Escherichia expression plasmid harboring six histidine residues fused to a pnrB gene was constructed (His6-PnrB) and designated pPSC1. The His6-PnrB induced in E. coli BL21 was purified using a nickel affinity column to homogeneity. Its enzymatic activity was assayed by measuring absorbance changes at 340 nm due to NADH oxidation. The V (max) and K ( m ) values of the enzyme for TNT were 12.6 micromol/min/mg protein and 2.9 mM, respectively. In addition, the pnrB knockout mutant was constructed via a single-crossover homologous recombination with a partial pnrB DNA fragment that lacked both start and stop codons. Eight days was required for complete degradation of 0.5 mM TNT by the wild-type HK-6 strain, whereas the pnrB mutant degraded only 10% of the TNT in the same time period. Even after 20 days, only approximately 50% of the 0.5 mM TNT was degraded by the pnrB mutant. These results illustrate that pnrB may perform a crucial role in the TNT degradation pathway of the HK-6 strain.

Highly Sensitive and Selective Determination of Manganese in Tea Leaves by a Catalytic Kinetic Spectrophotometric Method

AbstractThe catalytic effect of manganese(II) on the oxidation of barium diphenylamine sulfonate (BDAS) with potassium bromate in sulfuric acid was studied. Progress of the reaction was monitored, spectrophotometrically, by measuring absorbance changes at 547.5 nm. A highly sensitive, selective and simple method was accordingly developed for the determination of trace amounts of Mn(II), with no need for a separation or pre‐concentration step. Effects of reaction time, concentration of reagents, temperature and ionic strength on the reaction rate were studied. In the optimized conditions, manganese could be determined in a range of 1 to 60 ng mL−1 with a detection limit of 0.2 ng mL−1. The proposed method was successfully applied to the determination of manganese in tea leaves.

Spectroelectrochemical and electrochemical behavior of epinephrine at a gold electrode

Spectroelectrochemical and electrochemical behavior of epinephrine, adrenaline, at a gold electrode was studied with cyclic voltammetric and spectroelectrochemical techniques. Based on the analysis of a series of spectra obtained by in-situ spectroscopic measurements absorption bands of possible intermediates generated in the electrochemical oxidation of adrenaline could be assigned. Also, assignments of various peaks in the cyclicvoltammograms obtained in different pH conditions are not always in agreement with the past findings, especially the formation of dehydrated form of leucoadrenochrome is not likely to occur under acidic conditions. From the analysis of current voltage curves and simultaneously obtained absorption UV-Vis. spectra, it was able to assign the absorption characteristics of species generated. Adrenalinequinone ( λ=380 nm) is formed in the first step of oxidation of adrenaline ( λ=290 nm) followed by cyclization of the quinone product to produce leucoadrenochrome ( λ=310 nm). The leucoadrenochrome is finally converted into adrenochrome ( λ=470∼525 nm) chemically or electrochemically. Especially, derivative cyclic voltabsorptograms (DCVA) of a species obtained by measuring absorbance change with potential scans at a selected wavelength provide a new information on the oxidation of adrenaline.

Evidence for involvement of a zymogen granule Na+/H+ exchanger in enzyme secretion from rat pancreatic acinar cells.

We have characterized a Na+/H+ exchanger in the membrane of isolated zymogen granules (ZG) from rat exocrine pancreas and investigated its role in secretagogue-induced enzyme secretion. ZG Na+/H+ exchanger activity was estimated by measuring Na+ or Li+ influx and consequent osmotic swelling and lysis of ZG incubated in Na- or Li-acetate. Alternatively, intragranule pH was investigated by measuring absorbance changes in ZG which had been preloaded with the weak base acridine orange. Na+- or Li+-dependent ZG lysis was enhanced by increasing inward to outward directed H+ gradients. Na+-dependent ZG lysis was not prevented by an inside-positive K+ diffusion potential generated by valinomycin which argues against parallel operation of separate electrogenic Na+ and H+ permeabilities and for coupled Na+/H+ exchange through an electroneutral carrier. Na+- and Li+-dependent ZG lysis was inhibited by EIPA (EC50 approximately 25 microM) and benzamil (EC50 approximately 100 microM), but only weakly by amiloride. Similarly, absorbance changes due to release of acridine orange from acidic granules into the medium were obtained with Na+ and Li+ salts only, and were inhibited by EIPA, suggesting the presence of a Na+/H+ exchanger in the membrane. Na+ dependent lysis of ZG was inhibited by 0.5 mm MgATP and MgATP-gamma-S by about 60% and 35%, respectively. Inhibition by MgATP was prevented by incubation of ZG with alkaline phosphatase (100 U/ml), or by the calmodulin antagonists calmidazolium (0.75 microM), trifluoperazine (100 microM) and W-7 (500 microM), suggesting that the ZG Na+/H+ exchanger is regulated by a ZG membrane-bound calmodulin-dependent protein kinase. Na+ dependence of secretagogue (CCK-OP)-stimulated amylase secretion was investigated in digitonin permeabilized rat pancreatic acini and was higher in acini incubated in Na+ containing buffer (30 mm NaCl/105 mm KCl buffer; 6.4 +/- 0.4% of total amylase above basal) compared to buffer without Na+ (0 mm NaCl/135 mm KCl buffer; 4.7 +/- 0.4% of total amylase above basal, P < 0.03). EIPA (50 microM) reduced CCK-OP-induced amylase secretion in Na+ containing buffer from 7.5 +/- 0.6% to 4.1 +/- 0.8% (P < 0.02). In the absence of Na+ in the buffer, CCK-OP-stimulated amylase release was not inhibited by 50 microM EIPA. The data suggest that an amiloride insensitive, EIPA inhibitable Na+/H+ exchanger is present in ZG membranes, which is stimulated by calmodulin antagonists and could be involved in secretagogue-induced enzyme secretion from rat pancreatic acini.

Shot-noise-limited detection of absorbance changes induced by subpicojoule laser pulses in optical pump-probe experiments

We describe a high-frequency modulation and detection system capable of measuring absorbance changes ΔA on the order of 10−6 in optical pump-probe experiments using submilliwatt laser powers from a mode-locked ultrafast laser. For probe beam powers ≥9 μW, the system offers nearly shot-noise-limited performance.

Hyperthermophiles: taking the heat and loving it.

Hyperthermophiles, a recently discovered group of microorganisms, are operationally defined as having an optimum growth temperature of at least 80°C and a maximum growth temperature of over 90°C. Most of the enzymes isolated so far from such organisms exhibit correspondingly enhanced thermostability. This property has been used both for investigation of fundamental biological questions of protein structure and stability, and for the development of technological applications that require protein stability at high temperatures. Examples of such applications of thermostable proteins include their use in biocatalysts, in various materials and in crystallization methods. The use of DNA polymerases from hyperthermophilic microorganisms in the polymerase chain reaction (PCR) is another of the more practical examples of the recent impact on biochemistry and molecular biology of enzymes isolated from hyperthermophilic organisms. To date, the maximum growth temperatures observed for a hyperthermophilic organism is about 110°C. Whether this is the upper limit for life is unknown; some workers estimate that the maximum growth temperature for, as yet, uncultured organisms may approach, or even exceed 150°C [1]. In any event, it is clear that the surface has barely been scratched in the study of extremely thermostable proteins from known hyperthermophiles. Two basic questions often come to mind when considering these enzymes: the practically motivated, how can I get some of these enzymes and the more fundamental question, how do these enzymes achieve extreme thermostability? Aspects of both of these issues are briefly discussed below. More detailed discussions of hyperthermophiles and their proteins may be found in review articles [2], [3], [4] and [5].

A light emitting diode microspectrophotometer: intracellular Ca 2+ measurements in isolated stretch receptor

A microspectrophotometer was designed to measure absorbance changes in single cells. The device utilizes sequentially activated light emitting diodes (LED) to provide different wave lengths of light. The instrument has the advantage of relative simplicity and less cost compared to other devices. The spectrophotometer was tested by measuring absorbance changes of the metallochromic Ca2+ indicator Arsenazo III (AIII) injected into the crayfish (Astacus astacus) stretch receptor. Under the conditions described the detection limit of the concentration of AIII was 0.05 mM and absorbance changes of 0.0005 can be reliably determined which correspond to a detection limit of 10-20 nM for free Ca2+ changes assuming a light path length of 0.003 cm and an apparent dissociation constant (KD) of 2 microM for the Ca(2+)-AIII complex. The upper frequency limit of the device is 3000 Hz. The absorbance measurements of AIII injected into the crayfish stretch receptor neurons revealed a Ca(i) of 375 +/- 177 nM (mean +/- SD: 14 cells). LiCl substituted for NaCl increased Ca(i) 45-100 nM in different cells, suggesting that a Na+ gradient is necessary for Ca2+ homeostasis in this receptor.

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

Secondary structure change of myoglobin induced by sodium dodecyl sulfate and its kinetic aspects

Conformational change of horse myoglobin induced by sodium dodecyl sulfate (NaDodSO 4) was studied mainly by measurements of circular dichroism (CD) and stopped-flow methods. The relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed CD spectrum of reference spectra of the corresponding structures to the experimentally obtained CD spectrum of the protein. The helical content of the native myoglobin was found to be 82%. In the presence of 0.6 m M NaDodSO 4, the helical content was decreased to 58% and the β-structure, which did not exist in the absence of the surfactant, was assumed to some extent above 0.3 m M NaDodSO 4. This conformational change was followed in the Soret band as well as at 288 nm by the stopped-flow spectrophotometry. The corresponding rate constants were determined by measuring absorbance changes with time at these wavelengths. The experiments indicated that the environmental change of the heme group occurs simultaneously with the destruction of the helix. The rate constants showed NaDodSO 4 concentration dependence similar to those of conformational changes in other proteins in the surfactant solution. The binding isotherm of NaDodSO 4 to myoglobin was obtained using high-performance liquid chromatography. The binding sites of this anionic surfactant were estimated to be 0.8 by the Scatchard plot. The conformational change in myoglobin induced by NaDodSO 4 was correlated with its amino acid sequence in which each amino acid residue was considered to be either helical or non-helical.

Optical recording of calcium and voltage transients following impulses in cell bodies and processes of identified leech neurons in culture

Optical methods were used to examine the spread of electrical potentials and the distribution and time course of calcium transients in individual identified nerve cells isolated from the leech. A photodiode array detected voltage transients by measuring absorbance changes of cells stained with the voltage-sensitive dye RH155 added to the bath. Calcium transients were recorded by measuring absorbance changes of the dye arsenazo III, which had been injected into the cells. In addition, Lucifer yellow was injected to outline the some and processes. Calcium changes resulting from individual action potentials were recorded from N, P, and Retzius cells without averaging. Signals from T cells and anterior pagoda (AP) cells were weaker but could be detected with averaging. These results are in accord with previous studies on calcium contributions to action potentials in these cells. For all cells, larger or wider action potentials gave bigger signals. Calcium changes from each of a train of action potentials were of equal amplitude, showing no sign of facilitation. Calcium transients from Retzius cells that had formed chemical synapses with P cells had properties similar to those of isolated cells. We were also able to detect responses from prolonged subthreshold depolarizations to -40 mV from a hyperpolarized membrane potential (-65 mV). These signals rose throughout the duration of the pulse (1-2 sec). With the photodiode array we mapped the distribution of the calcium signals. The amplitudes from each pixel are proportional to the amount of calcium entering that element in response to the stimulating pulse, if the simplifying assumption is made that the calcium buffering of the cytoplasm is uniform throughout the cell. The largest signals were detected over the axon stump left from the cell isolation procedure. Large signals were also detected from the soma. Weak signals were detected from the processes of some cells. From many Retzius cells, no signals at all were detected from the newly formed processes. Using the photodiode array, we also recorded voltage transients from the cells. Signals were recorded from all over the arborization of the neuron, with no obvious variation in time course, showing that the entire cell, including fine slender processes and broad growth cones, was essentially isopotential. Combining these observations with the measured distribution of calcium transients in the same cell suggests that the density of calcium channels in most cells is less in the outgrowing processes than in the soma or axon stump.