Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Regulation of cellular calcium metabolism and calcium transport by calcitonin.

Calcitonin was studied in isolated kidney cells and in isolated mitochondria. A concentration of 10 ng/ml of synthetic calcitonin increases the cellular accumulation of 45Ca and the total cell calcium. The mitochondrial pool is increased several-fold. Kinetic analysis of the data shows that although the total cellular exchangeable calcium pool is enlarged, calcium influx and efflux are significantly depressed by calcitonin. The absence of phosphate or the presence of inhibitors of mitochondrial calcium transport completely abolish the effects of the hormone. In isolated mitochondria, the hormone stimulates the active calcium uptake and depresses the extramitochondrial calcium activity. Calcitonin counteracts the effects of cyclic AMP which stimulates the release of calcium from mitochondria and increases the extramitochondrial calcium activity. These data indicate that cellular calcium homeostasis is controlled by the mitochondrial calcium turnover. They suggest that calcitomin regulates the cell calcium metabolism and inhibits the transcellular calcium transport by stimulating the rate of calcium uptake by mitochondria which depresses cytoplasmic calcium activity.     

cAMP-induced cytoskeleton rearrangement increases calcium transients through the enhancement of capacitative calcium entry

In this report we investigated the correlation between cell morphology and regulation of cytosolic calcium homeostasis. Type I astrocytes were differentiated to stellate process-bearing cells by a 100-min exposure to cAMP. Differentiation of cortical astrocytes increased the magnitude and duration of calcium transients elicited by phospholipase C-activating agents as measured by single cell Fura-2-based imaging. Calcium imaging showed differences in the spatial pattern of the response. In both differentiated and the control cells, the response originated in the periphery and gradually extended into the center of the cell. However, the elevation of cytosolic calcium concentration ([Ca(2+)](i)) was particularly evident within the processes and adjacent to the inner cell membrane of the differentiated astrocytes. In addition, differentiation significantly prolonged the duration of the [Ca(2+)](i) elevation. Potentiation of the calcium transients was mimicked by forskolin-induced differentiation and abolished by a specific protein kinase-A blocker. Conversely, the enhancement of the calcium transients was not mimicked by brief exposure to cAMP not causing morphological differentiation, and in PC12 cells that did not undergo morphological changes after 100 min of cAMP treatment. Impairing cAMP-induced cytoskeleton re-organization, by means of cytochalasin D and nocodazole, prevented the potentiation of the calcium transients in cAMP-treated astrocytes. Phospholipase C activity and sensitivity to inositol (1,4,5)-trisphosphate were not involved in the enhancement of the calcium responses. Also, potentiation of the calcium transients was dependent on extracellular calcium. Calcium storage and thapsigargin-depletable intracellular calcium reservoirs were analogously not increased in differentiated astrocytes. Rearrangement of the cell shape also caused a condensation of the endoplasmic reticulum and altered the spatial relationship between the endoplasmic reticulum and the cell membrane. In conclusion, morphological rearrangements of type I astrocytes increase the magnitude and the duration of agonist-induced calcium transients via enhancement of capacitative calcium entry and is associated with a spatial reorganization of the relationship between cell membrane and the endoplasmic reticulum structures.     

Capacitative calcium entry: sensing the calcium stores

A long-standing mystery in the cell biology of calcium channel regulation is the nature of the signal linking intracellular calcium stores to plasma membrane capacitative calcium entry channels. An RNAi-based screen of selected Drosophila genes has revealed that a calcium-binding protein, stromal interaction molecule (STIM), plays an essential role in the activation of these channels and may be the long sought sensor of calcium store content.     

Cytosolic calcium oscillators

Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.     

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.     

Endoplasmic reticulum calcium pumps and cancer

Endoplasmic reticulum calcium homeostasis is involved in a multitude of signaling, as well as "house-keeping" functions that control cell growth, differentiation or apoptosis in every human/eukaryotic cell. Calcium is actively accumulated in the endoplasmic reticulum by Sarco/Endoplasmic Reticulum Calcium transport ATPases (SERCA enzymes). SERCA-dependent calcium transport is the only calcium uptake mechanism in this organelle, and therefore the regulation of SERCA function by the cell constitutes a key mechanism to adjust calcium homeostasis in the endoplasmic reticulum depending on the cell type and its state of differentiation. The direct pharmacological modulation of SERCA activity affects cell differentiation and survival. SERCA expression levels can undergo significant changes during cell differentiation or tumorigenesis, leading to modified endoplasmic reticulum calcium storage. In several cell types such as cells of hematopoietic origin or various epithelial cells, two SERCA genes (SERCA2 and SERCA3) are simultaneously expressed. Expression levels of SERCA3, a lower calcium affinity calcium pump are highly variable. In several cell systems SERCA3 expression is selectively induced during differentiation, whereas during tumorigenesis and blastic transformation SERCA3 expression is decreased. These observations point at the existence of a cross-talk, via the regulation of SERCA3 levels, between endoplasmic reticulum calcium homeostasis and the control of cell differentiation, and show that endoplasmic reticulum calcium homeostasis itself can undergo remodeling during differentiation. The investigation of the anomalies of endoplasmic reticulum differentiation in tumor and leukemia cells may be useful for a better understanding of the contribution of calcium signaling to the establishment of malignant phenotypes.     

Genetically encoded calcium indicators for studying long-term calcium dynamics during apoptosis

Intracellular calcium release is essential for regulating almost all cellular functions. Specific spatio-temporal patterns of cytosolic calcium elevations are critical determinants of cell fate in response to pro-apoptotic cellular stressors. As the apoptotic program can take hours or days, measurement of long-term calcium dynamics are essential for understanding the mechanistic role of calcium in apoptotic cell death. Due to the technical limitations of using calcium-sensitive dyes to measure cytosolic calcium little is known about long-term calcium dynamics in living cells after treatment with apoptosis-inducing drugs. Genetically encoded calcium indicators could potentially overcome some of the limitations of calcium-sensitive dyes. Here, we compared the performance of the genetically encoded calcium indicators GCaMP6s and GCaMP6f with the ratiometric dye Fura-2. GCaMP6s performed as well or better than Fura-2 in detecting agonist-induced calcium transients. We then examined the utility of GCaMP6s for continuously measuring apoptotic calcium release over the course of ten hours after treatment with staurosporine. We found that GCaMP6s was suitable for measuring apoptotic calcium release over long time courses and revealed significant heterogeneity in calcium release dynamics in individual cells challenged with staurosporine. Our results suggest GCaMP6s is an excellent indicator for monitoring long-term changes cytosolic calcium during apoptosis.     

Factors influencing calcium influx in endotoxin-challenged fibroblasts

The role of cell density and pH on calcium influx was studied in normal and endotoxin-challenged cultured 3T6 fibroblasts. In normal fibroblasts, at low cell densities, there was no marked difference in calcium influx at pH 6.6, 7.4, and 7.8, whereas at high cell densities, the calcium influx was markedly higher at pH 6.6 as compared to that at pH 7.8. Endotoxin treatment for 4 hr at low cell density and in alkaline pH (7.4-7.8) increased calcium influx in a dose-dependent manner. In contrast, at high cell density and low pH (6.6), endotoxin treatment markedly decreased calcium influx in a dose- and time-dependent manner. These endotoxin-induced changes in calcium influx were not fully compensated by altered calcium efflux because total calcium content of the cells was found to be altered. The efficacy of the endotoxin varied depending on the bacterial source of the endotoxin and the method of purification. There was a relationship between the effect of different endotoxins on the increase in calcium influx and the inhibition of cell proliferation. Endotoxin did not decrease, but slightly increased cell proliferation when added to high cell density cultures even at a concentration of 200 micrograms/ml.     

Disease-causing mutations of calcium channels

Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca2+ ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca2+ stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores. Mutations of genes encoding calcium have been implicated in the etiology of a diverse group of nerve and muscle diseases. These mutations have been identified in humans, mice and other organisms. In this review, we will summarize calcium channelopathies of humans and mice. Of the ten calcium channel α1 subunits cloned and sequenced (see ref. 1), disease-causing mutations have been found in CaV1.4 and CaV2.1 in the nervous system, and CaV1.1 and CaV1.2 in muscle. Mutations in calcium channel auxiliary subunits (α2δ, β and γ) have also been associated with both human and/or mouse neurological diseases. The disease-causing mutations may provide new insight into the cell biological roles of calcium channels as well as into relationships between structure and function. In addition, understanding how the mutations affect the physiology of the cell could lead to advances in disease treatment by relieving symptoms or slowing the progression of the disease. However, due to the multifaceted functions of calcium in the cell, the correlation between molecular mutation, physiological alterations and disease etiology is neither straightforward nor easily understood. Since calcium is an important intracellular signaling molecule, altered calcium channel function can give rise to widespread changes in cellular function. Indeed, serious diseases result from mutations that cause trivial alterations of calcium currents analyzed in vitro.     

Mitochondria: The calcium connection

Calcium handling by mitochondria is a key feature in cell life. It is involved in energy production for cell activity, in buffering and shaping cytosolic calcium rises and also in determining cell fate by triggering or preventing apoptosis. Both mitochondria and the mechanisms involved in the control of calcium homeostasis have been extensively studied, but they still provide researchers with long-standing or even new challenges. Technical improvements in the tools employed for the investigation of calcium dynamics have been–and are still–opening new perspectives in this field, and more prominently for mitochondria. In this review we present a state-of-the-art toolkit for calcium measurements, with major emphasis on the advantages of genetically encoded indicators. These indicators can be efficiently and selectively targeted to specific cellular sub-compartments, allowing previously unavailable high-definition calcium dynamic studies. We also summarize the main features of cellular and, in more detail, mitochondrial calcium handling, especially focusing on the latest breakthroughs in the field, such as the recent direct characterization of the calcium microdomains that occur on the mitochondrial surface upon cellular stimulation. Additionally, we provide a major example of the key role played by calcium in patho-physiology by briefly describing the extensively reported–albeit highly controversial–alterations of calcium homeostasis in Alzheimer's disease, casting lights on the possible alterations in mitochondrial calcium handling in this pathology.     

Cellular calcium and atherosclerosis: A brief review

Evidence for and against the theory that cell calcium is causally involved in the pathogenesis of atherosclerosis is presented and evaluated. In particular, it is argued that: (1) arterial calcium is increased in atherosclerosis; (2) this increase in tissue calcium content is largely intracellular; (3) this increased intracellular calcium content is caused by increased plasma membrane calcium permeability; (4) the increased calcium content is causally related to atherogenesis; (5) many of the cell physiological, cell biological, biochemical, and molecular biological processes, known to function abnormally in atherosclerosis, are also known to be calcium regulated; and (6) these processes are activated or inactivated in atherosclerosis in a manner consistent with increased cell calcium. It is concluded that the calcium-atherogenesis hypothesis has the potential to unify macroscopic clinical risk factors in terms of intracellular mechanisms that are controlled by cell calcium, and that this hypothesis deserves further experimental tests.     

Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium

Integrins are ubiquitous trans-membrane adhesion molecules that mediate the interaction of cells with the extracellular matrix (ECM). Integrins link cells to the ECM by interacting with the cell cytoskeleton. In cases such as leukocyte binding, integrins mediate cell-cell interactions and cell-ECM interactions. Recent research indicates that integrins also function as signal transduction receptors, triggering a number of intracellular signaling pathways that regulate cell behavior and development. A number of integrins are known to stimulate changes in intracellular calcium levels, resulting in integrin activation. Although changes in intracellular calcium regulate a vast number of cellular functions, this review will discuss the stimulation of calcium signaling by integrins and the role of intracellular calcium in the regulation of integrin-mediated adhesion.     

Mutual antagonism of calcium entry by capacitative and arachidonic acid-mediated calcium entry pathways

In nonexcitable cells, the predominant mechanism for regulated entry of Ca(2+) is capacitative calcium entry, whereby depletion of intracellular Ca(2+) stores signals the activation of plasma membrane calcium channels. A number of other regulated Ca(2+) entry pathways occur in specific cell types, however, and it is not know to what degree the different pathways interact when present in the same cell. In this study, we have examined the interaction between capacitative calcium entry and arachidonic acid-activated calcium entry, which co-exist in HEK293 cells. These two pathways exhibit mutual antagonism. That is, capacitative calcium entry is potently inhibited by arachidonic acid, and arachidonic acid-activated entry is inhibited by the pre-activation of capacitative calcium entry with thapsigargin. In the latter case, the inhibition does not seem to result from a direct action of thapsigargin, inhibition of endoplasmic reticulum Ca(2+) pumps, depletion of Ca(2+) stores, or entry of Ca(2+) through capacitative calcium entry channels. Rather, it seems that a discrete step in the pathway signaling capacitative calcium entry interacts with and inhibits the arachidonic acid pathway. The findings reveal a novel process of mutual antagonism between two distinct calcium entry pathways. This mutual antagonism may provide an important protective mechanism for the cell, guarding against toxic Ca(2+) overload.     

Interplay between mitochondria and cellular calcium signalling

Mitochondria are increasingly ascribed central roles in vital cell signalling cascades. These organelles are now recognised as initiators and transducers of a range of cell signals, including those central to activation and amplification of apoptotic cell death. Moreover, as the main source of cellular ATP, mitochondria must be responsive to fluctuating energy demands of the cell. As local and global fluctuations in calcium concentration are ubiquitous in eukaryotic cells and are the common factor in a dizzying array of intra- and inter-cellular signalling cascades, the relationships between mitochondrial function and calcium transients is currently a subject of intense scrutiny. It is clear that mitochondria not only act as local calcium buffers, thus shaping spatiotemporal aspects of cytosolic calcium signals, but that they also respond to calcium uptake by upregulating the tricarboxylic acid cycle, thus reacting metabolically to local signalling. In this chapter we review current knowledge of mechanisms of mitochondrial calcium uptake and release and discuss the consequences of mitochondrial calcium handling for cell function, particularly in conjunction with mitochondrial oxidative stress.     

Muscarinic receptor-induced calcium responses in astroglia

BACKGROUND: The objective of this study was to characterize and quantitate the calcium responses to cholinergic stimulation in individual primary rat cortical astrocytes and human 132 1N1 astrocytoma cells. Materials and Methods The fluorescent calcium probe Indo-1 AM and an attached cell analysis and sorting (ACAS) instrument were used to quantitate calcium responses in these cells. RESULTS: A concentration-dependent response to carbachol was seen in both cell types. However, carbachol was more potent and efficacious, and the response was more homogeneous in the cell line. The calcium response was mediated by the M3 subtype of muscarinic receptors. Experiments in the absence of extracellular calcium and with EGTA demonstrated that the initial calcium spike was due to calcium release from intracellular calcium stores, whereas the sustained elevation and oscillations were dependent on calcium influx. Protein kinase C exerts a feedback inhibition of these calcium responses, and appears to be involved in maintaining the elevated calcium concentration and oscillations. CONCLUSIONS: This study provided a detailed quantitation of the changes in intracellular calcium evoked in individual astroglial cells by activation of M3 muscarinic receptors. This will allow for the study of pharmacological and toxicological agents on this response.     

The B cell receptor-induced calcium flux involves a calcium mediated positive feedback loop

The B cell receptor (BCR)-elicited calcium flux results in activation of mature B cells. We have recently shown that the adaptor protein Swiprosin-1/EFhd2 (EFhd2) amplifies the BCR-induced calcium flux in B cell lines. EFhd2 is a calcium binding adaptor protein with two predicted EF-hands. Here we asked whether these domains are functional and control its function. Using a blot-overlay assay with radioactive calcium we show that both EF-hands of EFhd2 have an intrinsic capacity to bind calcium. Equilibrium centrifugation confirmed that EFhd2 binds 2 calcium ions, with an apparent Kd of 110 μM. Point mutations revealed that the conserved residues E116 and E152, which reside in the canonical calcium binding loop in EF-hands 1 and 2, are essential for calcium binding by EFhd2. These mutations as well as deletion of the EF-hands, in particular EF-hand 1, abolished the ability of EFhd2 to restore BCR-induced calcium signaling in EFhd2-deficient WEHI231 cells. N-terminal deletions, but not C-terminal deletions, acted similarly. Thus, the N-terminal part of EFhd2 as well as calcium binding to its EF-hands control the intracellular calcium concentration in response to BCR stimulation in WEHI231 cells. Hence, EFhd2 regulates the BCR-elicited calcium flux through a calcium-dependent positive feedback mechanism in WEHI231 cells.     

核钙信号

尽管核周隙与内质网的腔相通,核膜上存在钙信号分子的受体等事实表明,细胞核存在一套相对独立的钙信号机制。作为核钙的贮存库,核被是核钙信号的发源地。核被中钙离子的充盈状态影响着核孔复合体的构象,从而调节核质间物质交流。已有证据显示,核钙信号与胞质钙信号在基因转录中的作用有所区别。核钙信号在细胞凋亡中发挥重要作用,其中,钙蛋白酶起着较为关键的作用。核钙信号研究为完整理解钙信号的生理功能开辟了新视野。     

Store-operated calcium entry and increased endothelial cell permeability

We hypothesized that myosin light chain kinase (MLCK) links calcium release to activation of store-operated calcium entry, which is important for control of the endothelial cell barrier. Acute inhibition of MLCK caused calcium release from inositol trisphosphate-sensitive calcium stores and prevented subsequent activation of store-operated calcium entry by thapsigargin, suggesting that MLCK serves as an important mechanism linking store depletion to activation of membrane calcium channels. Moreover, in voltage-clamped single rat pulmonary artery endothelial cells, thapsigargin activated an inward calcium current that was abolished by MLCK inhibition. F-actin disruption activated a calcium current, and F-actin stabilization eliminated the thapsigargin-induced current. Thapsigargin increased endothelial cell permeability in the presence, but not in the absence, of extracellular calcium, indicating the importance of calcium entry in decreasing barrier function. Although MLCK inhibition prevented thapsigargin from stimulating calcium entry, it did not prevent thapsigargin from increasing permeability. Rather, inhibition of MLCK activity increased permeability that was especially prominent in low extracellular calcium. In conclusion, MLCK links store depletion to activation of a store-operated calcium entry channel. However, inhibition of calcium entry by MLCK is not sufficient to prevent thapsigargin from increasing endothelial cell permeability.     

Localization and quantitation of calcium pools and calcium binding sites in cultured human keratinocytes

Calcium plays a crucial role in regulating the growth and differentiation of cultured keratinocytes. However, the mechanism(s) of this regulation is not clear. Prior studies have shown that intracellular free calcium (Cai) increases with keratinocyte differentiation. In this study, in order to evaluate the role of cytosolic free calcium and organelle-bound calcium in keratinocyte differentiation, we quantitated and localized calcium pools in keratinocytes, utilizing the fluorescence probe indo-1 and ion-capture cytochemistry, respectively. Cai of undifferentiated keratinocytes was 80–120 nM, whereas Cai of differentiated keratinocytes was 200–300 nM depending on the extent of differentiation. The Cai of individual cells in an undifferentiated colony was heterogeneous (60–160 nM) with larger cells displaying higher Cai. Heterogeneity also was observed in the intracellular calcium-containing precipitates in the different layers of stratifying keratinocyte cultures using the cytochemical technique. Calcium precipitates were abundant in the lower cell layers, progressively decreasing apically, with the uppermost layer devoid of precipitates. Calcium-containing precipitates appeared as fine-tocoarse electron-dense granules on the plasma membrane, within the cytosol, mitochondria, nucleus, and vacuolar organelles. Whereas ionomycin in the presence of extracellular calcium increased the amount of intracellular calcium precipitates, EGTA removed calcium precipitates from organelles. Unlike intact epidermis, keratinocytes displayed no extracellular calcium reservoirs. Putative calcium binding sites, visualized by trivalent lanthanum (La) binding, were abundant on cell membranes and desmosomes of basaloid cells, but decreased in the upper cell layers. These studies revealed differences in the distribution of free ionic calcium (as determined by the fluorescence technique) and organelle-bound calcium (as determined by the cytochemical technique). Striking differences were also observed in calcium localization between intact epidermis and cultured epidermal cells. The localization pattern of calcium in cultured keratinocytes may reflect the hyperproliferative state of these cells, as in psoriatic epidermis, and/or the absence of a normal permeability barrier in these submerged cultures. © 1993 Wiley-Liss, Inc.