Are Cav1.3 pacemaker channels in chromaffin cells? Possible bias from resting cell conditions and DHP blockers usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca²⁺ channels expressed in these cells (Ca_v1.2 and  Ca_v1.3). Using  Ca_v1.3^-/- KO MCCs we have shown that  Ca_v1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV)¹: low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds.  Ca_v1.2 contributes also to pacemaking MCCs and possibly even Na⁺ channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (–50 mV),  Ca_v1.3 carries equal amounts of Ca²⁺ current to  Ca_v1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Nav1 channels that activate at 24 mV more positive potentials than  Ca_v1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is:  Ca_v1.3,  Ca_v1.2 and Na_v1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching “non-firing” into “firing” cells that may lead to erroneous conclusions about the role of Ca_v1.3 and  Ca_v1.2 as pacemaker channels in MCCs.²     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca (2+) -dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (CaV1.2 and CaV1.3), CaV1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using CaV1 .3 (-/-) KO mice and the AP-clamp it has been possible to resolve the time course of CaV1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells CaV1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca (2+) currents and activate BK channels confers to CaV1.3 the unique feature of driving Ca (2+) loading during long interspike intervals and, possibly, to control the Ca (2+) -dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

CaV1.3 as pacemaker channels in adrenal chromaffin cells: Specific role on exo- and endocytosis?

Voltage-gated L-type calcium channels (LTCCs) are expressed in adrenal chromaffin cells. Besides shaping the action potential (AP), LTCCs are involved in the excitation-secretion coupling controlling catecholamine release and in Ca²⁺-dependent vesicle retrieval. Of the two LTCCs expressed in chromaffin cells (Ca_V1.2 and Ca_V1.3), Ca_V1.3 possesses the prerequisites for pacemaking spontaneously firing cells: low-threshold, steep voltage-dependence of activation and slow inactivation. By using Ca_V1 .3^-/- KO mice and the AP-clamp it has been possible to resolve the time course of Ca_V1.3 pacemaker currents, which is similar to that regulating substantia nigra dopaminergic neurons. In mouse chromaffin cells Ca_V1.3 is coupled to fast-inactivating BK channels within membrane nanodomains and controls AP repolarization. The ability to carry subthreshold Ca²⁺ currents and activate BK channels confers to Ca_V1.3 the unique feature of driving Ca²⁺ loading during long interspike intervals and, possibly, to control the Ca²⁺-dependent exocytosis and endocytosis processes that regulate catecholamine secretion and vesicle recycling.     

Regulated exocytosis in chromaffin cells and cytotoxic T lymphocytes: How similar are they?

Chromaffin cells from the adrenal medulla secrete catecholamines into the blood stream as part of the fight-or-flight response. Cytotoxic T lymphocytes from the immune system release cytotoxic substances to kill antigen-presenting cells. While at first glance these two cell types do not seem to have much in common, evidence from human diseases indicates that the molecular mechanisms of exocytosis of the respective granules share many similar features. In this review we highlight the similarities and differences of individual aspects of granule maturation and release in both cell types. In addition, we discuss established and putative molecules involved in distinct steps and suggest technical approaches which might facilitate future studies in chromaffin cells and cytotoxic T lymphocytes.     

Restricted T cell receptor V-β and J-β usage in T cells from interleukin-2-cultured lymphocytes of ovarian and renal carcinomas

Tumour-infiltrating lymphocytes (TIL) are often observed in human tumours and their presence has been correlated with a better prognosis. It has been suggested that TIL are enriched for tumour-specific cytotoxic cells, and TIL activated and expanded in vitro by interleukin-2 (IL-2) are currently used in the therapy of human cancer. We have studied the T cell repertoire in IL-2-expanded TIL cells from patients with ovarian and renal carcinoma using T-cell-receptor-V--specific monoclonal antibodies and a polymerase-chain-reaction-based Southern blot technique for analysis of J- usage. In TIL lines derived from three of nine patients with ovarian carcinomas and from two of eight patients with renal carcinomas, selective usage of the V-6 or V-5 T-cell receptor gene products was found. The majority of the cells were CD4⁺, with up to 40% of the T cells utilizing the same V- gene. T-cell lines derived from peripheral blood lymphocytes from patients or healthy donors contained normal levels of V- subsets. Only moderate levels of V-6⁺ T cells were detected from freshly isolated TIL and the increase of this subpopulation appeared as a result of in vitro culture. The level of clonal restriction, as measured by the usage of J- gene segments within the V-5 or V-6 families, was analysed using a recently developed technique based on the polymerase chain reaction. Evidence for restricted J- usage was detected only in TIL expanded in vitro, while this was not the case in freshly isolated tumour-derived lymphocytes or T cell lines obtained from peripheral blood lymphocytes. The presence of a population with biased T cell receptor expression in cells derived from tumour tissue could be explained by their activation in vivo as a result of contact with tumour antigens and should be taken into consideration when discussing the therapeutic efficiency of IL-2-expanded TIL.     

Mitochondrial disappearance from cells: a clue to the role of autophagy in programmed cell death and disease?

When cells are induced to undergo apoptosis in the presence of general caspase inhibitors and then returned to their normal growth environment, there follows an extended period of life during which the entire cohort of mitochondria (including mitochondrial DNA) disappears from the cells. This phenomenon is widespread; it occurs in NGF-deprived sympathetic neurons, in NGF-maintained neurons treated with cytosine arabinoside, and in diverse cell lines treated with staurosporine, including HeLa, CHO, 3T3 and Rat 1 cells. Mitochondrial removal is highly selective since the structure of all other organelles remains unperturbed. Since Bcl2 overexpression blocks the removal of mitochondria without preventing death-inducing signals, it appears that the mitochondria are responsible for initiating their own demise. Degradation of mitochondria is not in itself a rare event. It occurs in large part by autophagy during normal cell house-keeping, during ecdysis in insects, as well as after induction of apoptosis. However, the complete and selective removal of an entire cohort of mitochondria in otherwise living mammalian cells has not been described previously. These findings raise several questions. What are the mechanisms which remove mitochondria in such a 'clean' fashion? What are the signals that target mitochondria for such selective degradation? How are cells that have lost their mitochondria different from rho0 cells (which retain mitochondria but lack mitochondrial DNA, and cannot carry out oxidative phosphorylation)? Are the cells which have lost mitochondria absolutely committed to die or might they be repaired by mitochondrial therapy? The answers will be especially relevant when considering treatment of diseases affecting long-lived and non-renewable organs such as the nervous system.     

Are polyphenols antioxidants or pro-oxidants? What do we learn from cell culture and in vivo studies?

Diets rich in polyphenols are epidemiologically associated with lower risk of developing some age-related diseases in humans. This apparent disease-protective effect of polyphenols is often attributed to their powerful antioxidant activities, as established in vitro. However, polyphenols can also exert pro-oxidant activities under certain experimental conditions. Neither pro-oxidant nor anti-oxidant activities have yet been clearly established to occur in vivo in humans, nor are they likely given the limited levels of polyphenols that are achievable in vivo after consumption of foods and beverages rich in them. Other actions of polyphenols may be more important in vivo. Many studies of the biological effects of polyphenols in cell culture have been affected by their ability to oxidise in culture media, and awareness of this problem can avoid erroneous claims.     

Apoptosis and cell death in neuronal cells: where does Ca2+ fit in?

Mounting evidence shows that neuronal death is an important and essential component of brain tissue homeostasis, with major forms of cell death occurring: necrosis and apoptosis. No general consensus exists as to whether these two forms of neuronal death represent separate cellular processes or just two different forms of a common 'death pathway'. One difference between them is the role played by intracellular Ca2+: central and obligatory, in necrosis and possible, but not always necessary in triggering apoptosis. Furthermore, the same assessment of the involvement of Ca2+ signalling could also distinguish between two possible apoptotic states in the nervous system: one, the 'developmental apoptosis', involving immature and developing neurons, in which Ca2+ plays mainly an apoprotector role, and another one, associated mainly with pathological instances and involving fully matured and established neurons, in which Ca2+ plays an apo-inducing role.     

Cell–cell junction remodeling in the heart: Possible role in cardiac conduction system function and arrhythmias?

Anchoring cell-cell junctions (desmosomes, fascia adherens) play crucial roles in maintaining mechanical integrity of cardiac muscle cells and tissue. Genetic mutations and/or loss of critical components in these macromolecular structures are increasingly being associated with arrhythmogenic cardiomyopathies; however, their specific roles have been primarily attributed to effects within the working (ventricular) cardiac muscle. Growing evidence also points to a key role for anchoring cell-cell junction components in cardiac muscle cells of the cardiac conduction system. This is not only evidenced by the molecular and ultra-structural presence of anchoring cell junctions in specific compartments/structures of the cardiac conduction system (sinoatrial node, atrioventricular node, His-Purkinje system), but also because conduction system-related arrhythmias can be found in humans and mouse models of cardiomyopathies harboring defects and/or mutations in key anchoring cell-cell junction proteins. These studies emphasize the clinical need to understand the molecular and cellular role(s) for anchoring cell-cell junctions in cardiac conduction system function and arrhythmias. This review will focus on (i) experimental findings that underline an important role for anchoring cell-cell junctions in the cardiac conduction system, (ii) insights regarding involvement of these structures in age-related cardiac remodeling of the conduction system, (iii) summarizing available genetic mouse models that can target cardiac conduction system structures and (iv) implications of these findings on future therapies for arrhythmogenic heart diseases.     

New frontiers in human cell biology and medicine: Can pluripotent stem cells deliver?

Human pluripotent stem cells provide enormous opportunities to treat disease using cell therapy. But human stem cells can also drive biomedical and cell biological discoveries in a human model system, which can be directly linked to understanding disease or developing new therapies. Finally, rigorous scientific studies of these cells can and should inform the many science and medical policy issues that confront the translation of these technologies to medicine. In this paper, I discuss these issues using amyotrophic lateral sclerosis as an example.Much of modern cell biological discovery has been driven by the study of a diverse variety of primary and transformed cultured cells. However, the advent of human pluripotent cells is providing new avenues of discovery. These cells are genetically manipulable, euploid, expandable to large numbers, and can differentiate to most if not all human cell types. In addition, these cells can be used to analyze the large number of mutations and diverse genetic variation present in large human populations. In fact, not only are human pluripotent stem cells useful for typical cell culture experiments, but they are amenable to many of the types of genetic and molecular genetic approaches that historically have only been feasible in genetic and developmental systems, such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, or mouse.There are two types of human pluripotent stem cells in use. Human embryonic stem cells (hESC) are derived from human embryos that would otherwise be discarded and that are generally donated with substantial informed consent and ethical requirements (Shamblott et al., 1998; Thomson et al., 1998). Human induced pluripotent stem cells (hIPSC) are generated by several different reprogramming technologies, generally from fibroblasts obtained from small skin biopsies or other human somatic cell types, such as blood (Takahashi et al., 2007). Recent work suggests that hESC and hIPSC, although not identical in their properties, share very important features. First, hESC and hIPSC are both pluripotent so that any cell type of interest can in principle be generated. In fact, differentiation methods for many types of specialized human cells are being developed rapidly, fueled in large part by the need to generate stable differentiated derivatives for cell therapy. Second, hIPSC and hESC can be handled in the laboratory under conditions that are relatively straightforward for skilled cell culture scientists. Third, both hIPSC and hESC, when handled properly, are genetically relatively stable with a diploid karyotype so that gene dose and gene expression at all loci is effectively “normal” or at least representative of expression levels in cells in the intact human. These properties make these cells or their differentiated derivatives suitable for genetic screens using RNA interference, small molecules, insertional mutagenesis, or other analogous tools. Important differences between hESC and hIPSC include an apparent elevation in mutation load in hIPSC (Gore et al., 2011) and differences in epigenetic state. Either of these features may substantially influence the behavior of each cell type and its differentiated derivatives. Thus, hESC and hIPSC may play different roles in the discovery of new cellular and disease mechanisms and in the development of cellular therapies. Recent examples of novel discoveries made using hESC and hIPSC include substantial new insights into the control of the pluripotent state itself and identification of molecular pathways controlling cellular differentiation.

Disease in a dish approaches with hESC and hIPSC

Although hESC and hIPSC are just beginning to be used to probe and elucidate new cellular processes, there is already substantial progress using pluripotent stem cell approaches to unravel disease mechanisms using so-called disease in a dish paradigms (Unternaehrer and Daley, 2011). Disease in a dish methods use gene manipulation and/or reprogramming technologies to generate hESC or hIPSC lines with genomes carrying known mutations causing human disease, lesions such as shRNA expression mimicking human disease mutations (Marchetto et al., 2010; Ordonez et al., 2012), or genomes carrying combinations of known or unknown variants that contribute to disease (Fig. 1). With the advent of powerful molecular tools, such as Tal effector nucleases, individual genes can be manipulated by introducing point mutations with great precision (Hockemeyer et al., 2011). Thus, disease-causing mutations or other genetic lesions can be studied for their impacts on cellular processes under “normal” conditions of gene expression and in different genetic backgrounds. Similarly, suppressor and enhancer studies are feasible and will help unravel poorly understood cellular mechanisms. Finally, after differentiation to specialized cell types, cellular mechanisms and interactions as well as disease and potential therapies can be evaluated in bona fide human cells. These approaches are in their infancy but have substantial potential given the limitations of mouse models of disease to accurately recapitulate human disease and the many obvious differences between the details of mouse physiology and human physiology. They also bring unique advantages for diseases in which the key cell types, e.g., human central nervous system neurons, are difficult if not impossible to obtain in good condition or early in disease.Open in a separate windowFigure 1.Disease in a dish. Stem cells can be used to analyze how human genetic variation or mutation contributes to defined cellular phenotypes. Using neuronal phenotype as an example, Tal effector nucleases (TALENs) can be used to make defined changes in hIPSC of a common genetic background to analyze the contribution of a defined mutation or more complex variation to neuronal phenotypes.Examples of recent disease in dish studies include a variety of neurodegenerative diseases, heart diseases, and diseases of other organ systems (Unternaehrer and Daley, 2011). Although this work is in its early stages, there is a great deal of potential for meaningful mechanistic cell biological research in this collection of intriguing areas. In addition, these disease in dish models provide unique human materials for direct testing of drug safety and efficacy.

Amyotrophic lateral sclerosis (ALS): A disease in a dish paradigm

ALS, also known as Lou Gehrig’s disease, provides an intriguing example that illustrates the path from mechanism-based research to understanding and potential therapies using disease in a dish approaches. ALS is a disease in which death of motor neurons causes paralysis of voluntary muscles. As the disease progresses, paralysis ultimately extends to the muscles involved in breathing, swallowing, and all other voluntary movements. Once diagnosed, ALS generally causes death within 3–5 yr or less. There is only one approved drug for ALS, riluzole, but individual patients do not perceive much benefit because riluzole generates statistical prolongation of life for only a few months based on large-scale clinical trials. The key problem of course is to learn what causes death of motor neurons in ALS and whether this information might help develop a therapy that protects motor neurons from dysfunction and death.Although most ALS is “sporadic,” some forms are hereditary, including a form caused by mutations in the gene encoding dismutase SOD1 (superoxide 1). The generation of transgenic mouse and rat models that carry human mutant SOD1 genes has led to substantial progress in the understanding of cellular mechanisms that contribute to disease. In particular, a series of genetic studies in transgenic and chimeric mice led to the realization that the death of motor neurons in ALS might not be cell autonomous (Clement et al., 2003; Boillée et al., 2006; Yamanaka et al., 2008a,b). Disease in a dish studies using astrocytes carrying SOD1 mutant genes mixed with in vitro differentiated motor neurons made from pluripotent stem cells confirmed these findings and lead directly to searches for secreted toxic factors and drug testing (Di Giorgio et al., 2007, 2008; Marchetto et al., 2008). The general conclusion from these studies is that motor neuron death in ALS is strongly influenced by other cells in the spinal cord that make important contributions to, or protect from, motor neuron death. Whether astrocytes, microglia, or other cell types carry mutant SOD1 genes determines whether they exhibit stimulatory or protective effects on motor neuron death. Although such conclusions might be limited to SOD1-mediated ALS, there is also recent evidence that astrocytes might contribute to sporadic ALS as well (Haidet-Phillips et al., 2011).

Development of cell replacement or augmentation therapies

Successfully treating debilitating and currently incurable diseases with cell replacement or augmentation therapies requires basic cell biological research to fuel the generation and testing of new therapies (Fig. 2). For example, expansion of hematopoietic stem cell therapeutic approaches from leukemias to other diseases is based on a sound understanding of the basic cell and developmental biology of these cells. Several different stem cell therapies are in the midst of development and testing for several disorders, including spinal cord injury, graft versus host disease, skin diseases, blindness, diabetes, and AIDS (e.g., California Institute for Regenerative Medicine, 2009; Pollack, 2012; Schwartz et al., 2012). Using ALS as an example, some stem cell–based therapy efforts are aimed at trying to replace motor neurons that are damaged or die in ALS. But inducing motor neurons or their stem cell precursors to engraft into spinal cords or upper motor cortex and then appropriately extend axons and wire to peripheral muscles may be a challenge that will take many years to solve. Interestingly, the evidence that ALS is non–cell autonomous with major contributions from astrocytes and other glia has led to two different categories of cell therapy approach. One approach, which I regard as little more than guesswork, has tried to treat ALS by introducing poorly defined mesenchymal stem cells or cord blood stem cells directly into the spinal cord of ALS model animals. In fact, even in the absence of strong and reliable evidence, a clinical trial of cord blood stem cells transplanted into the spinal cord of human ALS patients was launched by a private company (http://www.tcacellulartherapy.com/fda_clinical_trials.html) and then halted by the FDA. A more rational approach given the state of scientific understanding, the state of experiments in animal models, and the in vitro data is to introduce progenitor cells that can differentiate to astrocytes or progenitors that secrete growth factors (Klein et al., 2005; Suzuki et al., 2007; Lepore et al., 2008; Suzuki and Svendsen, 2008; Hefferan et al., 2012). One of these approaches has recently reached clinical trials using fetal-derived spinal cord stem cells in which one hopes that enough will be learned to support more trials using different stem cell–generated preparations and perhaps different surgical methods or spinal cord sites.Open in a separate windowFigure 2.Cell replacement therapy using stem cells. A possible path from patients with hereditary ALS to cell biological research using transgenic mouse or pluripotent stem cell models to cell therapies. In this example, assessing the contribution of different cell types to ALS through rigorous research can lead to a rational approach to cell therapy.

Driving evidence-based scientific and medical policy with stem cell–driven discovery

The social and medical issues that arise in the development of cell therapies are and will be heavily influenced by the scientific discoveries about and using human stem cells. These social and medical challenges are well illustrated by a discussion of ALS owing to its rapidly progressive and devastating nature.First is whether one type of therapy can treat all types of ALS patients. Solving this issue will require a better understanding of what causes ALS, what cellular mechanisms contribute to motor neuron death, and which cells contribute in different forms of ALS. One key and possibly false assumption that drives current efforts is that all forms of ALS will exhibit the type of cellular nonautonomy found in animal models of SOD1-mediated ALS. Thus, models of sporadic ALS and hereditary forms of ALS such as those mediated by FUS/TLS or TDP-43 mutations must be tested. These experiments will also allow tests of the magnitude of the relative contributions of different cell types to motor neuron death or rescue in different forms of ALS. Additionally, if the actual cellular pathways that are defective in astrocytes and motor neurons can be better defined, cellular augmentation strategies and drug discovery could take advantage of that information.Second is the so-called snake oil problem (http://www.closerlookatstemcells.org; CBSNews, 2010). Numerous misleading and probably fraudulent advertisements can be found about clinics offering stem cell cures for ALS. These wild claims ignore large amounts of scientific data about the nature of ALS and rational approaches to therapy and prey upon those who don’t have ready access to or cannot evaluate legitimate scientific and medical information. Our legitimate scientific and medical community needs to stand against these frauds and provide accurate information derived from rigorous research to patients so that they are not taken advantage of by these clinics. In addition, we must work to ensure that legitimate efforts are not damaged by the blowback from those who effectively steal from desperately ill patients and their families or the likely harm to these patients that is coming from clinics that dispense untested and sometimes dangerous therapies.Third is the cell tracking problem. Currently, it is difficult to know how cells transplanted into the spinal cord of an ALS patient behave until after a patient has died. In addition, using antibodies to examine postmortem material from a transplant patient is problematic because the transplants are of human cells into a human patient. We desperately need to develop safe and sensitive methods for cell marking and imaging that will allow us to track cell behavior in patients in real time after transplant. Real-time measures would allow therapy to be modified or even repeated based on the analysis of cell behavior. These methods will rely heavily on cell biological research to identify cellular pathways and markers that could be visualized in real time by magnetic resonance imaging, positron emission tomography, or other imaging modalities.Fourth is how to manage individual patient response versus the average response of patients in a clinical trial. Although most often thought about with respect to drug therapies, different forms of ALS might vary in their response to cell therapies. An interesting possibility is that hIPSC lines from individual patients could be used not only for drug testing but also for evaluating the genetically driven contribution of different cell types to each patient’s version of ALS. A corollary is that for ALS patients included in a clinical trial, the notion that cells would be introduced only once and that the patient would then be “passively” followed with no change in treatment paradigm until death might be unacceptable. In conventional medicine, one might try treatment again or modify treatment course, depending on how an individual patient responds. On the other hand, prospective design of a statistically rigorous clinical trial requires development of a treatment plan and identification of rigorous outcome measures that should not be modified if the data are to be interpretable. Development of new statistical methods, outcome measures, hIPSC evaluation of phenotypes, and perhaps, cellular marking methods might allow trials to tolerate modification as part of an ALS patient’s clinical care. Perhaps rigorous data from hIPSC-based research could be used to make a case to the FDA that ALS clinical trials need to be more responsive to patient needs and variable outcomes with a disease that is as complicated and clinically inconsistent as ALS.Fifth, and finally, is the risk benefit analysis that can hinder or accelerate the development of therapies for rapidly fatal diseases such as ALS. Current paradigms of therapy development are risk averse and require enormous amounts of information on safety and possible efficacy before trials can be approved, financed, and launched. Yet, some patient populations, such as those with ALS, when facing a near certain death sentence, are very risk tolerant and might be willing to participate in trials with much lower certainty. Our community must work with the FDA to tackle this problem and to perhaps dramatically accelerate the introduction of good, but perhaps radical, ideas that might work but in which safety or efficacy testing in animals could take many years, or simply be unreliable, so that current patients would have no hope of benefiting. I often ask myself, as I work with my colleagues to develop a cell-based therapy for ALS that has been partially tested in animals but is not yet complete and therefore not ready for humans, what I would do if I, my wife, or one of my children developed ALS. Would I be willing to have appropriate types of stem cells or their derivatives transplanted into them even if I were not absolutely certain and had not yet proven absolute safety or efficacy? Interestingly, I find that in thinking about this issue, I fall back on my scientific understanding of ALS and the rigorous types of data on ALS mechanisms in the mainstream scientific literature. The result is that my own risk tolerance rises substantially when I have the ability to consider published and unpublished data and how it might be applied. I think that all ALS patients should have this information and that the FDA should be responsive to these patients when they want to take well-informed risks with experimental therapies that may not yet meet current FDA standards. Clearly, the devil will be in the details for implementing such an approach and ensuring patient protection as well as opportunity, but we owe this consideration to current ALS patients and those with comparably severe diseases. Again, however, this is a debate in which rigorous scientific research can drive the agenda and resulting policies.

Concluding remarks

Virchow developed the concept that disease arises in the individual cells of a tissue (Schultz, 2008). This important principle is the foundation for using human stem cells to understand basic cellular mechanisms and to extend that understanding to the development of therapies. Treating disease by targeting the misbehaving cells is clearly a wonderful opportunity for therapy development and research. Thus, probing the secrets of human cells by taking advantage of human pluripotent stem cells may signal the dawn of a new era in cell biology research.Finally, consider the many remarkable discoveries and novel mechanisms found when the basic tools of cell and molecular biology were applied to unusual members of the model organism toolbox, including snakes, ciliates, planaria, jerboa, and other organisms that have developed unusual biological strategies during evolution. Could humans be added to this list, and could the study of basic human cell biology yield comparable discoveries? Because humans are a large, long-lived organism with a complex brain, a rich evolutionary history, and substantial genetic variation across large and accessible populations, the answer is certain to be yes.     

Liquid-like chromatin in the cell: What can we learn from imaging and computational modeling?

Chromatin in eukaryotic cells is a negatively charged long polymer consisting of DNA, histones, and various associated proteins. With its highly charged and heterogeneous nature, chromatin structure varies greatly depending on various factors (e.g. chemical modifications and protein enrichment) and the surrounding environment (e.g. cations): from a 10-nm fiber, a folded 30-nm fiber, to chromatin condensates/droplets. Recent advanced imaging has observed that chromatin exhibits a dynamic liquid-like behavior and undergoes structural variations within the cell. Current computational modeling has made it possible to reconstruct the liquid-like chromatin in the cell by dealing with a number of nucleosomes on multiscale levels and has become a powerful technique to inspect the molecular mechanisms giving rise to the observed behavior, which imaging methods cannot do on their own. Based on new findings from both imaging and modeling studies, we discuss the dynamic aspect of chromatin in living cells and its functional relevance.     

Are they still viable? Physical conditions and abundance of <Emphasis Type="Italic">Daphnia pulicaria</Emphasis> resting eggs in sediment cores from lakes in the Tatra Mountains

All species of Daphnia (Cladocera) produce, at some stage in their life cycle, diapausing eggs, which can remain viable for decades or centuries forming a “seed bank” in lake sediments. Because of their often good preservation in lake sediment, they are useful in paleolimnology and microevolutionary studies. The focus of this study was the analysis of cladoceran resting eggs stored in the sediment in order to examine the ephippial eggs bank of Daphnia pulicaria Forbes in six mountain lakes in the High Tatra Mountains, the Western Carpathians (northern Slovakia and southern Poland). Firstly, we analyzed distribution, abundance and physical condition of resting eggs in the sediment for their later used in historical reconstruction of Daphnia populations by genetic methods. To assess changes in the genetic composition of the population through time, we used two microsatellite markers. Although DNA from resting eggs preserved in the High Tatra Mountain lake sediments was extracted by various protocols modified for small amounts of ancient DNA, DNA from eggs was not of sufficient quality for microsatellite analyses. Distribution curves of resting eggs from sediment cores correspond to the environmental changes that have occurred in the High Tatra Mountains area during last two centuries (atmospheric acid deposition, fish introduction) and demonstrate their influence on natural populations. Evaluation of ephippia physical condition (the most common category was empty ephippial covers) suggests that the majority of resting eggs hatched to produce a new generation of Daphnia or may be due to failed deposition of resting eggs by Daphnia to the chitinous case. In conclusion, age, low quantity and poor physical condition of resting eggs from these Tatra lake sediments proved to be unsuitable not just for use in genetic analyses, but also the possibilities of autogenous restoration of Daphnia populations from the resting egg banks in the Tatra sediments are negligible.     

Differences in mortality from acute myocardial infarction between coronary care unit and medical ward: treatment or bias?

To analyse the effectiveness of coronary care units in reducing mortality from myocardial infarction 18 hospitals ranging from large urban teaching hospitals to small country hospitals were stratified into four levels of care. Previous analysis had failed to show significant differences in the overall mortality in hospital among levels. There were significant differences in mortality, however, between those patients allocated to be cared for in the coronary care unit and those in the medical wards in the more advanced hospitals. The differences were largest in the hospitals with the most elaborate facilities (level 1) and non-existent in those with the least (level 4). Several analytical approaches to these observed differences indicated that they were: (a) reduced by adjustment for age and severity of infarction; (b) paralleled by differences in coexisting disease recorded on death certificates; (c) no longer significant at level 1 after allowing for differences in coexisting disease; and (d) not significant at any level after exclusion of patients first diagnosed at necropsy. These findings suggest that the observed differences in mortality between coronary care units and medical wards are largely due to bias in selection and diagnosis.     

A Highlights from MBoC Selection: MgcRacGAP restricts active RhoA at the cytokinetic furrow and both RhoA and Rac1 at cell–cell junctions in epithelial cells

Localized activation of Rho GTPases is essential for multiple cellular functions, including cytokinesis and formation and maintenance of cell–cell junctions. Although MgcRacGAP (Mgc) is required for spatially confined RhoA-GTP at the equatorial cortex of dividing cells, both the target specificity of Mgc''s GAP activity and the involvement of phosphorylation of Mgc at Ser-386 are controversial. In addition, Mgc''s function at cell–cell junctions remains unclear. Here, using gastrula-stage Xenopus laevis embryos as a model system, we examine Mgc''s role in regulating localized RhoA-GTP and Rac1-GTP in the intact vertebrate epithelium. We show that Mgc''s GAP activity spatially restricts accumulation of both RhoA-GTP and Rac1-GTP in epithelial cells—RhoA at the cleavage furrow and RhoA and Rac1 at cell–cell junctions. Phosphorylation at Ser-386 does not switch the specificity of Mgc''s GAP activity and is not required for successful cytokinesis. Furthermore, Mgc regulates adherens junction but not tight junction structure, and the ability to regulate adherens junctions is dependent on GAP activity and signaling via the RhoA pathway. Together these results indicate that Mgc''s GAP activity down-regulates the active populations of RhoA and Rac1 at localized regions of epithelial cells and is necessary for successful cytokinesis and cell–cell junction structure.     

Light and norepinephrine similarly prevent damping of the melatonin rhythm in cultured chick pineal cells: regulation of coupling between the pacemaker and overt rhythms?

The circadian rhythm of melatonin output displayed by chick pineal cells in static culture damps rapidly in constant red light (RR). This can be seen in the first cycle following a switch from a cycle of 12 hr white light (L) and 12 hr red light (R) to RR. Melatonin output is higher during the "day" in R than it is in L, but higher that next night (in R) after daytime L than after daytime R. This effect might be due entirely to the entraining effect of L. Alternatively, the higher nocturnal output after daytime L could be related to the acute suppression caused by L; it might be a "rebound" phenomenon. These alternative hypotheses differ in their predictions for the effects of norepinephrine (NE) and pertussis toxin (PT). Previous results dissociated the acute and entraining effects of L: PT blocks the acute effect but not the entraining effect of L. NE mimics the acute effect of L (and is blocked by PT), but not the entraining effect. If L prevents damping entirely by entrainment, then NE should not mimic and PT should not block this same-cycle effect of daytime L on nocturnal melatonin output. However, the present research found that NE did mimic and PT did block this effect, indicating that the ability of L to prevent damping is mediated by a same-cycle "rebound" following L's acute inhibition of melatonin production. Furthermore, NE enhanced the "rebound" effect of daytime L, and cycles substituting NE for L were effective in driving the melatonin rhythm. Lowering extracellular potassium did not induce a "rebound," and adding exogenous melatonin did not prevent one. The difference between nocturnal melatonin synthesis after daytime R and that after daytime L or NE implies regulation of coupling between the output of the circadian pacemaker and melatonin production. These results also suggest a role for NE in regulating and maintaining the expression of the melatonin rhythm.     

2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) activity in cultured nerve cell lines from central nervous system: Comparison of proliferating and resting growth states and cell cycle-dependent activity changes

1. The present communication is concerned with the expression and cell cycle-dependent regulation of the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) in cultured nerve cell lines derived from the rat central nervous system (CNS). 2. The enzyme activity was measured in relation to two reversible serum-controlled growth states (exponentially growing/quiescent) including a comparison of the enzyme activities in cell lines of neuronal and glial origin as well as in fibroblasts. CNPase is present in all cell types tested, but the enzyme activity is very sensitive to changes in the cellular growth state. Nerve cell lines in exponentially growing cultures express a 3 to 15 times higher specific CNPase activity than the nonneural cell types. In serum-starved quiescent cultures, the differences in specific enzyme activity between the nerve cell lines and the fibroblasts are enlarged even more up to a ratio of about 50 to 150, indicating a specific function of this enzyme within the central nervous system. 3. Neuron-like B104 cells could be stimulated to synchronized growth by serum readdition to quiescent cultures. A series of ordered activity changes of CNPase has been observed after the reinitiation of cell growth. The enzyme is stimulated at two particular stages during the cell cycle, leading to a biphasic activity profile. Maximum stimulation of CNPase correlates with the G1 phase. 4. Hydroxyurea-induced blockage of synchronized B104 cells to traverse the S phase also prevents the subsequent stimulation of CNPase activity. Therefore, we conclude that a correlation exists between the periodic activity changes of CNPase and particular phases of the B104 cell cycle.     

Comparison of electroporation and Chariot? for delivery of ��-galactosidase into mammalian cells: strategies to use trehalose in cell preservation

There are many compounds that can and have been used as cryoprotectants including disaccharides such as trehalose. Many organisms in nature use trehalose to help protect themselves at colder temperatures. Trehalose has also been used to a limited extent for the preservation of mammalian cells and tissues, but mainly as a supplement to other cryoprotectants like dimethyl sulfoxide. Recently, the use of trehalose as the primary cryoprotectant has gained much interest because of its low-potential cytotoxicity. Trehalose does not readily pass through mammalian cells membranes and research has shown that it is most effective when present on both sides of the cell membrane prior to preservation. Different strategies for introducing disaccharide sugars into cells have been investigated with limited success. In this study, two separate strategies are investigated for the introduction of disaccharide sugars into cells. Electroporation using an electric pulse to create temporary holes in the membrane so that molecules could pass through and a transport peptide (Chariot?) that covalently binds to the molecule of interest and then moves it across the membrane. Both strategies have the potential to load disaccharide sugars into cells at concentrations that would provide ample protection during preservation. In preparation for cryopreservation studies, smooth muscle cells that are difficult to cryopreserve using conventional preservation protocols were used to evaluate and compare the translocation potential of these two strategies using β-galactosidase. Assessment of each loading strategy was done by measuring viability and the presence of β-galactosidase inside the cells. The results indicate that both methods appear feasible as potential delivery systems and that treatment cytotoxicity can be minimized. The next step is definition of the best loading strategy to introduce trehalose into cells followed by preservation by freezing.     

B cell subsets and dysfunction of regulatory B cells in IgG4-related diseases and primary Sj?gren’s syndrome: the similarities and differences

Introduction

IgG4-related disease (IgG4-RD) is a multisystem-involved autoimmune disease. Abnormally activated and differentiated B cells may play important roles. Regulatory B cells (Breg) are newly defined B cell subgroups with immunosuppressive functions. In this study, we investigated the differences of B cell subsets, the expressions of co-stimulatory molecules on B cells, and the function of Breg cells in patients with IgG4-RD, primary Sjögren’s syndrome (pSS) as well as in healthy controls (HC).

Methods

Newly diagnosed IgG4-RD patients (n = 48) were enrolled, 38 untreated pSS patients and 30 healthy volunteers were recruited as disease and healthy controls. To analyze B cell subsets and B cell activity, PBMCs were surface stained and detected by flow cytometry. The function of Breg cells was tested by coculturing isolated CD19 + CD24^hiCD38^hi Breg cells with purified CD4 + CD25- T cells. Serum cytokines were measured by ELISA and cytometric bead array. Relationship between clinical data and laboratory findings were analyzed as well.

Results

Compared with pSS patients and HC, IgG4-RD patients had a lower frequency of peripheral Breg cells. Interestingly, CD19 + CD24-CD38^hi B cell subsets were significantly higher in peripheral B cells from IgG4-RD patients than in pSS patients and HC, which correlated with serum IgG4 levels. The expression of BAFF-R and CD40 on B cells was significantly lower in IgG4-RD patients compared with those in pSS patients and HC. Unlike HC, Breg cells from pSS patients lacked suppressive functions.

Conclusions

B cells in patients with IgG4-RD and pSS display a variety of abnormalities, including disturbed B cell subpopulations, abnormal expression of key signaling molecules, co-stimulatory molecules, and inflammatory cytokines. In addition, a significantly increased B cell subset, CD19 + CD24-CD38^hi B cells, may play an important role in the pathogenesis of IgG4-RD.