High-dose chemotherapy supported by autologous stem cell transplantation (ASCT) is an effective treatment for patients with lymphomas. However, failure to reach the minimum threshold of hematopoietic stem cells to proceed to ASCT may occur, even with the most effective strategies currently available.
Fludarabine-based regimens are highly effective as first-line therapy in patients with follicular lymphoma. Nevertheless, noticeable haematological toxicity has been reported using fludarabine-based regimens.
We describe the composition and distribution of bivalve molluscs from the sandy and rocky intertidal and the shallow subtidal environments of Bahía de Mazatlán, México. The bivalve fauna of the bay is represented by 89 living species in 28 families, including 37 new records and four range extensions: Lithophaga hastasia, Adula soleniformis, Mactrellona subalata, and Strigilla ervilia. The number of species increases from the upper (44) and lower intertidal (53) to the shallow subtidal (76), but only 11 (17%) have a wide distribution in the bay (i.e., found in all sampling sites and environments). The bivalve assemblages are composed of four main life forms: 27 epifaunal species, 26 infaunal, 16 semi-infaunal, and 20 endolithic. A taxonomic distinctness analysis identified the sampling sites and environments that contribute the most to the taxonomic diversity (species to suborder categories) of the bay. The present work increased significantly (31%) to 132 species previous inventories of bivalves of Bahía de Mazatlán. These species represent 34% of the bivalve diversity of the southern Golfo de California and approximately 15% of the Eastern Tropical Pacific region.
Calcium sparks in cardiac myocytes are brief, localized calcium releases from the sarcoplasmic reticulum (SR) believed to be caused by locally regenerative calcium-induced calcium release (CICR) via couplons, clusters of ryanodine receptors (RyRs). How such regeneration is terminated is uncertain. We performed numerical simulations of an idealized stochastic model of spark production, assuming a RyR gating scheme with only two states (open and closed). Local depletion of calcium in the SR was inevitable during a spark, and this could terminate sparks by interrupting CICR, with or without assumed modulation of RyR gating by SR lumenal calcium. Spark termination by local SR depletion was not robust: under some conditions, sparks could be greatly and variably prolonged, terminating by stochastic attrition-a phenomenon we dub "spark metastability." Spark fluorescence rise time was not a good surrogate for the duration of calcium release. Using a highly simplified, deterministic model of the dynamics of a couplon, we show that spark metastability depends on the kinetic relationship of RyR gating and junctional SR refilling rates. The conditions for spark metastability resemble those produced by known mutations of RyR2 and CASQ2 that cause life-threatening triggered arrhythmias, and spark metastability may be mitigated by altering the kinetics of the RyR in a manner similar to the effects of drugs known to prevent those arrhythmias. The model was unable to explain the distributions of spark amplitudes and rise times seen in chemically skinned cat atrial myocytes, suggesting that such sparks may be more complex events involving heterogeneity of couplons or local propagation among sub-clusters of RyRs.
? Malignant hyperthermia (MH) is linked to mutations in the type 1 ryanodine receptor, RyR1, the Ca2+ channel of the sarcoplasmic reticulum (SR) of skeletal muscle. The Y522S MH mutation was studied for its complex presentation, which includes structurally and functionally altered cell cores. Imaging cytosolic and intra-SR [Ca2+] in muscle cells of heterozygous YS mice we determined Ca2+ release flux activated by clamp depolarization, permeability (P) of the SR membrane (ratio of flux and [Ca2+] gradient) and SR Ca2+ buffering power (B). In YS cells resting [Ca2+]SR was 45% of the value in normal littermates (WT). P was more than doubled, so that initial flux was normal. Measuring [Ca2+]SR(t) revealed dynamic changes in B(t). The alterations were similar to those caused by cytosolic BAPTA, which promotes release by hampering Ca2+-dependent inactivation (CDI). The [Ca2+] transients showed abnormal breaks, decaying phases after an initial rise, traced to a collapse in flux and P. Similar breaks occurred in WT myofibres with calsequestrin reduced by siRNA; calsequestrin content, however, was normal in YS muscle. Thus, the Y522S mutation causes greater openness of the RyR1, lowers resting [Ca2+]SR and alters SR Ca2+ buffering in a way that copies the functional instability observed upon reduction of calsequestrin content. The similarities with the effects of BAPTA suggest that the mutation, occurring near the cytosolic vestibule of the channel, reduces CDI as one of its primary effects. The unstable SR buffering, mimicked by silencing of calsequestrin, may help precipitate the loss of Ca2+ control that defines a fulminant MH event.
The popularity of ecotourism in the marine protected areas of Mexico has increased over the last 10 years; in particular there is a large development of a SCUBA diving industry in the Mexican Pacific including Isabel Island. Given the risks associated with human activity in the marine environments around this island, we propose two ecotourism management strategies: (1) the creation and use of underwater trails, and (2) the estimation of the specific tourism carrying capacity (TCC) for each trail. Six underwater trails were selected in sites that presented elements of biological, geological, and scenic interest, using information obtained during field observations. The methodology used to estimate the TCC was based upon the physical and biological conditions of each site, the infrastructure and equipment available, and the characteristics of the service providers and the administrators of the park. Correction factors of the TCC included elements of the quality of the visit and the threat and vulnerability of the marine environment of each trail (e.g., divers expertise, size and distance between groups of divers, accessibility, wind, coral coverage). The TCC values ranged between 1,252 and 1,642 dives/year/trail, with a total of 8,597 dives/year for all six trails. Although these numbers are higher than the actual number of recreational visitors to the island (~1,000 dives per year), there is a need for adequate preventive management if the diving sites are to maintain their esthetic appeal and biological characteristics. Such management might be initially directed toward using only the sites and the TCC proposed here.
Imaging, optical mapping, and optical multisite recording of transmembrane potential (V(m)) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in V(m). Accordingly, they have been used for monitoring V(m) in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER--the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands-was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast "slit scanner" increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.
Chronic lymphocytic leukemia is the most common chronic lymphoproliferative disorder in Spain. The clinical management of this entity varies widely. Currently, in Spain, there are no national consensus guidelines, such as those published in other countries, to guide the diagnosis and treatment of this malignancy and the use of prognostic scores. This article reviews the current scientific literature and addresses issues on the diagnosis of chronic lymphocytic leukemia, the spread of the disease, the presence of comorbidities, the classification of prognostic scores, the common treatment regimens stratified by risk factors, and the management of complications associated with both the disease and its treatment, as well as the various controversies related to this entity. This document was drafted with the collaboration of national experts and aims to establish practical guidelines with their corresponding levels of evidence and grades of recommendation to guide the diagnosis, treatment and follow-up of patients with chronic lymphocytic leukemia.
Recent research suggests that the diastolic ryanodine-receptor-mediated release of Ca(2+) (J(leak)) from the sarcoplasmic reticulum of ventricular myocytes occurs in spark and nonspark forms. Further information about the role(s) of these release manifestations is scarce, however. This study addresses whether the fraction of spark-mediated J(leak) increases due to ?-adrenergic stimulation. Confocal microscopy was used to simultaneously image Ca(2+) sparks and quantify J(leak) in intact rabbit myocytes, either in the absence or in the presence of 125 nM isoproterenol. It was found that isoproterenol treatment shifts the spark-frequency-J(leak) relationship toward an increased sensitivity to a [Ca(2+)] trigger. In agreement, a small but significant increase in spark width was found for cells with matched baseline [Ca(2+)] and total SR [Ca(2+)]. The reconstruction of release fluxes, when applied to the average sparks from those selected cells, yielded a wider release source in the isoproterenol event, indicating the recruitment of peripheral ryanodine receptors. Overall, the results presented here indicate that ?-adrenergic stimulation increases the spark-dependent fraction of J(leak). Working together, the increased Ca(2+) sensitivity and the greater spark width found during isoproterenol treatment may increase the probability of Ca(2+) wave generation.
We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca(2+)] at equilibrium and an approximate analytic expression that does not require the equilibrium assumption. The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark.
Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.
The mechanisms that terminate Ca(2+) release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca(2+) concentration inside the sarcoplasmic reticulum (SR), [Ca(2+)](SR), simultaneously with that in the cytosol, [Ca(2+)](c), during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca(2+) release flux (derived from [Ca(2+)](c)(t)) over the gradient that drives it (essentially equal to [Ca(2+)](SR)) provided directly, for the first time, a dynamic measure of the permeability to Ca(2+) of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca(2+)](SR) stabilized at ?35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ?10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca(2+) release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca(2+)](SR), sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca(2+) release.
Current fluorescent monitors of free [Ca(2+)] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca(2+). "D4cpv-Casq1," expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective K(D) of 222 µM and a dynamic range [(R(max) - R(min))/R(min)] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, R(max), and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca(2+)](SR) in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca(2+)](SR) was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca(2+) concentration.
We evaluated the cost-effectiveness of rituximab added to the chemotherapy regimen of fludarabine plus cyclophosphamide (R-FC) versus fludarabine plus cyclophosphamide (FC) for the treatment of patients with previously untreated or relapsed/refractory chronic lymphocytic leukemia (CLL).
The aim of this study was to assess the incidence of cytomegalovirus (CMV) infection and disease in patients with hematologic malignancies treated with alemtuzumab. The outcome of CMV infection in hematologic patients treated with alemtuzumab in 19 hospitals throughout Spain was assessed retrospectively. Data were collected from the medical records of patients over a period of 6 months following initiation of alemtuzumab therapy. We studied 102 patients (89 with B-cell chronic lymphocytic leukemia and 13 with other lymphoproliferative diseases, with a median age of 63 years [range 29-81 years]). Alemtuzumab was administered for a mean of 11.2 (standard deviation: 13.8) weeks, with a median total dose of 423 mg (range: 59-1440 mg). Alemtuzumab as a single agent was administered in 92.2% of patients and was associated with chemotherapy in 7.8% of cases. Prophylactic antivirals included famcyclovir (47%), acyclovir (34%), valacyclovir (14%) and valgancyclovir (5%). CMV viremia testing was performed a mean of 6.3 times (range: 1-19). The incidence of CMV infection was 38.9% (46% in patients treated with steroids and 75% in patients receiving ?1000 mg of alemtuzumab). Treatment of CMV infection included gancyclovir or valgancyclovir in 94% of cases. Viremia became negative after a median of 20 days (95% CI: 13.4-26.6). CMV disease occurred in five patients. The incidence of CMV infection in alemtuzumab-treated patients was 38.9%. The incidence increased in patients treated concomitantly with steroids and in those treated with high doses of alemtuzumab, although only eight patients received 1000 mg or more, systematic monitoring of CMV viremia and early treatment of infection resulted in a favorable outcome of CMV reactivation.
We evaluated the clinical results of lenalidomide (Len) as a compassionate salvage therapy in refractory/relapsed multiple myeloma (MM) patients. A nationwide multi-centre, retrospective research study was performed to evaluate clinical data from patients with advanced MM for which compassionate use of lenalidomide was requested. The primary endpoints were the overall response rate (ORR) and the time to progression (TTP). Secondary objectives included safety and overall survival (OS) since starting of lenalidomide therapy. Data collected from the Spanish Compassionate Use Registry included 111 patients treated in 2006-2008. The median (range) number of previous treatment lines was 3 (1-8). The median duration of lenalidomide treatment while on study was of 4.9 months (1-18). Dexamethasone was given concomitantly with Len in 89% of patients. The ORR was 66% (4% of patients had stringent complete, 11% complete and 11% very good partial responses). Median TTP and OS were 13.0 and 17.4 months, respectively. The depth of response was significantly associated with a longer OS. Toxicity, mainly myelosuppression, was predictable and manageable with Len dose adjustments and cytokine support. Lenalidomide as salvage compassionate therapy in refractory/relapsed MM showed, in this series of heavily pre-treated patients, similar efficacy to that reported in pivotal clinical trials with acceptable tolerance.
The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (??m), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage.
Contractile activation in striated muscles requires a Ca(2+) reservoir of large capacity inside the sarcoplasmic reticulum (SR), presumably the protein calsequestrin. The buffering power of calsequestrin in vitro has a paradoxical dependence on [Ca(2+)] that should be valuable for function. Here, we demonstrate that this dependence is present in living cells. Ca(2+) signals elicited by membrane depolarization under voltage clamp were compared in single skeletal fibers of wild-type (WT) and double (d) Casq-null mice, which lack both calsequestrin isoforms. In nulls, Ca(2+) release started normally, but the store depleted much more rapidly than in the WT. This deficit was reflected in the evolution of SR evacuability, E, which is directly proportional to SR Ca(2+) permeability and inversely to its Ca(2+) buffering power, B. In WT mice E starts low and increases progressively as the SR is depleted. In dCasq-nulls, E started high and decreased upon Ca(2+) depletion. An elevated E in nulls is consistent with the decrease in B expected upon deletion of calsequestrin. The different value and time course of E in cells without calsequestrin indicate that the normal evolution of E reflects loss of B upon SR Ca(2+) depletion. Decrement of B upon SR depletion was supported further. When SR calcium was reduced by exposure to low extracellular [Ca(2+)], release kinetics in the WT became similar to that in the dCasq-null. E became much higher, similar to that of null cells. These results indicate that calsequestrin not only stores Ca(2+), but also varies its affinity in ways that progressively increase the ability of the store to deliver Ca(2+) as it becomes depleted, a novel feedback mechanism of potentially valuable functional implications. The study revealed a surprisingly modest loss of Ca(2+) storage capacity in null cells, which may reflect concurrent changes, rather than detract from the physiological importance of calsequestrin.
Growth hormone receptor (GHR) is a critical regulator of growth and metabolism. Although two GHRs have been characterized in many fish species, their functional characteristics, mechanisms of regulation and roles in embryonic development remain unclear. The zebrafish (Danio rerio) is an excellent model organism to study both developmental and physiological processes. In the present work, we characterized the complete cDNA sequences of zebrafish GHRs, ghra and ghrb, and their gene structures. We studied the expression of both receptors in adult tissues, and during embryonic development and larval stages by means of RT-PCR and whole-mount in situ hybridization. We determined that both transcripts are maternal ones, with specific expression patterns during development. Both GHR transcripts are mainly expressed in the notochord, myotomes, anterior structures and in the yolk cell. Interestingly, their expression became undetectable at 96h post-fertilization. Unlike other reports in fish, ghrs expression could not be detected in brain when adult tissues were used, and we detected ghrb but not ghra transcripts in muscle. In addition, we determined alternative transcript sequences for ghra with specific domain deletions, and alternative transcripts for ghrb that generate a premature stop codon and codify for truncated isoforms. These isoforms lack intracellular regions necessary for the activation of signal transducers and activators of transcription (STAT) family transcription factors 5.
Diastolic Ca leak from the sarcoplasmic reticulum (SR) of ventricular myocytes reduces the SR Ca content, stabilizing the activity of the SR Ca release channel ryanodine receptor for the next beat. SR Ca leak has been visualized globally using whole-cell fluorescence, or locally using confocal microscopy, but never both ways. When using confocal microscopy, leak is imaged as "Ca sparks," which are fluorescent objects generated by the local reaction-diffusion of released Ca and cytosolic indicator. Here, we used confocal microscopy and simultaneously measured the global ryanodine-receptor-mediated leak rate (J(leak)) and Ca sparks in intact mouse ventricular myocytes. We found that spark frequency and J(leak) are correlated, as expected if both are manifestations of a common phenomenon. However, we also found that sparks explain approximately half of J(leak). Our strategy unmasks the presence of a subresolution (i.e., nonspark) release of potential physiological relevance.
Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by degeneration of motor neurons and atrophy of skeletal muscle. Mutations in the superoxide dismutase (SOD1) gene are linked to 20% cases of inherited ALS. Mitochondrial dysfunction has been implicated in the pathogenic process, but how it contributes to muscle degeneration of ALS is not known. Here we identify a specific deficit in the cellular physiology of skeletal muscle derived from an ALS mouse model (G93A) with transgenic overexpression of the human SOD1(G93A) mutant. The G93A skeletal muscle fibers display localized loss of mitochondrial inner membrane potential in fiber segments near the neuromuscular junction. These defects occur in young G93A mice prior to disease onset. Fiber segments with depolarized mitochondria show greater osmotic stress-induced Ca(2+) release activity, which can include propagating Ca(2+) waves. These Ca(2+) waves are confined to regions of depolarized mitochondria and stop propagating shortly upon entering the regions of normal, polarized mitochondria. Uncoupling of mitochondrial membrane potential with FCCP or inhibition of mitochondrial Ca(2+) uptake by Ru360 lead to cell-wide propagation of such Ca(2+) release events. Our data reveal that mitochondria regulate Ca(2+) signaling in skeletal muscle, and loss of this capacity may contribute to the progression of muscle atrophy in ALS.
Phagocytosis of microbial invaders represents a fundamental defense mechanism of the innate immune system. The subsequent killing of microbes is initiated by the respiratory burst, in which nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates vast amounts of superoxide anion, precursor to bactericidal reactive oxygen species. Cytoplasmic pH regulation is crucial because NADPH oxidase functions optimally at neutral pH, yet produces enormous quantities of protons. We monitored pH(i) in individual human neutrophils during phagocytosis of opsonized zymosan, using confocal imaging of the pH sensing dye SNARF-1, enhanced by shifted excitation and emission ratioing, or SEER. Despite long-standing dogma that Na(+)/H(+) antiport regulates pH during the phagocyte respiratory burst, we show here that voltage-gated proton channels are the first transporter to respond. During the initial phagocytotic event, pH(i) decreased sharply, and recovery required both Na(+)/H(+) antiport and proton current. Inhibiting myeloperoxidase attenuated the acidification, suggesting that diffusion of HOCl into the cytosol comprises a substantial acid load. Inhibiting proton channels with Zn(2+) resulted in profound acidification to levels that inhibit NADPH oxidase. The pH changes accompanying phagocytosis in bone marrow phagocytes from HVCN1-deficient mice mirrored those in control mouse cells treated with Zn(2+). Both the rate and extent of acidification in HVCN1-deficient cells were twice larger than in control cells. In summary, acid extrusion by proton channels is essential to the production of reactive oxygen species during phagocytosis.
Many molecular biological interventions in current use, as well as inheritable disease conditions, modify the intracellular endowment of molecules that bind Ca(2+) or channels and pumps that transfer it to and from intracellular storage organelles. A simple law, named the "cell boundary theorem," states that intracellular alterations cannot directly result in changes in the cytosolic concentration, [Ca(2+)](i), in a true resting state. A demonstration of the validity of this theorem is provided. Several examples are then discussed of interventions or diseases that increase leak of Ca(2+) from storage organelles and result in greater resting [Ca(2+)](i). According to the theorem, the increase in [Ca(2+)](i) cannot be a direct consequence of the greater leak. Its primary cause must be a change of the fluxes at the level of the plasmalemma, caused in turn by the increase in leak through some sort of "store-operated Ca(2+) entry." While the law is discussed in terms of Ca(2+) homeostasis, it applies to any solute that may be transported by the plasma membrane.
Since its discovery in 1971, calsequestrin has been recognized as the main Ca(2+) binding protein inside the sarcoplasmic reticulum (SR), the organelle that stores and upon demand mobilizes Ca(2+) for contractile activation of muscle. This article reviews the potential roles of calsequestrin in excitation-contraction coupling of skeletal muscle. It first considers the quantitative demands for a structure that binds Ca(2+) inside the SR in view of the amounts of the ion that must be mobilized to elicit muscle contraction. It briefly discusses existing evidence, largely gathered in cardiac muscle, of two roles for calsequestrin: as Ca(2+) reservoir and as modulator of the activity of Ca(2+) release channels, and then considers the results of an incipient body of work that manipulates the cellular endowment of calsequestrin. The observations include evidence that both the Ca(2+) buffering capacity of calsequestrin in solution and that of the SR in intact cells decay as the free Ca(2+) concentration is lowered. Together with puzzling observations of increase of Ca(2+) inside the SR, in cells or vesicular fractions, upon activation of Ca(2+) release, this is interpreted as evidence that the Ca(2+) buffering in the SR is non-linear, and is optimized for support of Ca(2+) release at the physiological levels of SR Ca(2+) concentration. Such non-linearity of buffering is qualitatively explained by a speculation that puts together ideas first proposed by others. The speculation pictures calsequestrin polymers as wires that both bind Ca(2+) and efficiently deliver it near the release channels. In spite of the kinetic changes, the functional studies reveal that cells devoid of calsequestrin are still capable of releasing large amounts of Ca(2+) into the myoplasm, consistent with the long term viability and apparent good health of mice engineered for calsequestrin ablation. The experiments therefore suggest that other molecules are capable of providing sites for reversible binding of large amounts of Ca(2+) inside the sarcoplasmic reticulum.
The buffering power, B, of the sarcoplasmic reticulum (SR), ratio of the changes in total and free [Ca(2+)], was determined in fast-twitch mouse muscle cells subjected to depleting membrane depolarization. Changes in total SR [Ca(2+)] were measured integrating Ca(2+) release flux, determined with a cytosolic [Ca(2+)] monitor. Free [Ca(2+)](SR) was measured using the cameleon D4cpv-Casq1. In 34 wild-type (WT) cells average B during the depolarization (ON phase) was 157 (SEM 26), implying that of 157 ions released, 156 were bound inside the SR. B was significantly greater when BAPTA, which increases release flux, was present in the cytosol. B was greater early in the pulse - when flux was greatest - than at its end, and greater in the ON than in the OFF. In 29 Casq1-null cells, B was 40 (3.6). The difference suggests that 75% of the releasable calcium is normally bound to calsequestrin. In the nulls the difference in B between ON and OFF was less than in the WT but still significant. This difference and the associated decay in B during the ON were not artifacts of a slow SR monitor, as they were also found in the WT when [Ca(2+)](SR) was tracked with the fast dye fluo-5N. The calcium buffering power, binding capacity and non-linear binding properties of the SR measured here could be accounted for by calsequestrin at the concentration present in mammalian muscle, provided that its properties were substantially different from those found in solution. Its affinity should be higher, or K(D) lower than the conventionally accepted 1 mm; its cooperativity (n in a Hill fit) should be higher and the stoichiometry of binding should be at the higher end of the values derived in solution. The reduction in B during release might reflect changes in calsequestrin conformation upon calcium loss.
Splenic marginal zone lymphoma (SMZL) is a rare B-cell malignancy, with no standard treatment other than splenectomy. Rituximab has shown encouraging results. We therefore retrospectively assessed 43 patients from two centres, who received rituximab, either alone or with chemotherapy. All patients responded, 34/43 (79%) achieving a complete response (CR), compared with 3/10 (30%) after chemotherapy without rituximab (P = 0·005). Of these 10 patients, 9 (90%) subsequently achieved a CR after rituximab (P = 0·02). Rituximab monotherapy appeared equally as effective as rituximab combination therapy (90% vs. 79% CR, P = 0·7) with significantly less toxicity (12·5% vs. 83%, P = 0·002). Splenectomized patients were more likely to obtain a CR with rituximab (16/16, 100%) than unsplenectomized patients (18/27, 67%, P = 0·008). Disease-free survival (DFS) at 3 years was better after rituximab than after splenectomy alone [79% (95% confidence interval 60-89) vs. 29% (8-54), Hazard ratio (HR) 0·28 (0·12-0·68), P = 0·003] and better than after chemotherapy without rituximab [25% (4-55), HR 0·21 (0·08-0·51), P = 0·0004]. Survival at 3 years after rituximab was 98%. In summary, the CR and DFS rates after rituximab, given alone or with chemotherapy, were significantly better than after chemotherapy without rituximab in the same patients, with manageable toxicity. Rituximab, with or without splenectomy, should be considered for the treatment of SMZL.
Parameters (amplitude, width, kinetics) of Ca(2+) sparks imaged confocally are affected by errors when the spark source is not in focus. To identify sparks that were in focus, we used fast scanning (LSM 5 LIVE; Carl Zeiss) combined with fast piezoelectric focusing to acquire x-y images in three planes at 1-µm separation (x-y-z-t mode). In 3,000 x-y scans in each of 34 membrane-permeabilized cat atrial cardiomyocytes, 6,906 sparks were detected. 767 sparks were in focus. They had greater amplitude, but their spatial width and rise time were similar compared with all sparks recorded. Their distribution of amplitudes had a mode at ?F/F(0) = 0.7. The Ca(2+) release current underlying in-focus sparks was 11 pA, requiring 20 to 30 open channels, a number at the high end of earlier estimates. Spark frequency was greater than in earlier imaging studies of permeabilized ventricular cells, suggesting a greater susceptibility to excitation, which could have functional relevance for atrial cells. Ca(2+) release flux peaked earlier than the time of peak fluorescence and then decayed, consistent with significant sarcoplasmic reticulum (SR) depletion. The evolution of fluorescence and release flux were strikingly similar for in-focus sparks of different rise time (T). Spark termination involves both depletion of Ca(2+) in the SR and channel closure, which may be synchronized by depletion. The observation of similar flux in sparks of different T requires either that channel closure and other termination processes be independent of the determinants of flux (including [Ca(2+)](SR)) or that different channel clusters respond to [Ca(2+)](SR) with different sensitivity.
The contribution of Ca2+-induced Ca2+ release (CICR) to trigger muscle contraction is controversial. It was studied on isolated muscle fibres using synthetic localized increases in Ca2+ concentration, SLICs, generated by two-photon photorelease from nitrodibenzofuran (NDBF)-EGTA just outside the permeabilized plasma membrane. SLICs provided a way to increase cytosolic [Ca2+] rapidly and reversibly, up to 8 ?M, levels similar to those reached during physiological activity. They improve over previous paradigms in rate of rise, locality and reproducibility. Use of NDBF-EGTA allowed for the separate modification of resting [Ca2+], trigger [Ca2+] and resting [Mg2+]. In frog muscle, SLICs elicited propagated responses that had the characteristics of CICR. The threshold [Ca2+] for triggering a response was 0.5 ?M or less. As this value is much lower than concentrations prevailing near channels during normal activity, the result supports participation of CICR in the physiological control of contraction in amphibian muscle. As SLICs were applied outside cells, the primary stimulus was Ca2+, rather than the radiation or subproducts of photorelease. Therefore the responses qualify as ‘classic CICR. By contrast, mouse muscle fibres did not respond unless channel-opening drugs were present at substantial concentrations, an observation contrary to the physiological involvement of CICR in mammalian excitation–contraction coupling. In mouse muscle, the propagating wave had a substantially lower release flux, which together with a much higher threshold justified the absence of response when drugs were not present. The differences in flux and threshold may be ascribed to the absence of ryanodine receptor 3 (RyR3) isoforms in adult mammalian muscle.
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