Reserve Compartment Research Articles

A simple DEB-based ecosystem model.

A minimum stoichiometric carbon and nitrogen model of an entire ecosystem based on Dynamic Energy Budget (DEB) theory is presented. The ecosystem contains nutrients, producers, consumers, decomposers and detritus. All three living groups consist of somatic structure and either one (consumers and decomposers) or two (producers) reserve compartments, hence the living matter is described by seven state variables. Four types of detritus are distinguished. As the system is closed for matter, the dynamics of the nutrients carbon dioxide and ammonium follow automatically from the dynamics of the other 11 state variables. All DEB organisms in the model are V1-morphs, which means that surface area of each organism is proportional to volume. The resulting ontogenetic symmetry implies that complicated modelling of size structure is not required. The DEB V1-morph model is explained in detail, and the same holds for the idea of synthesizing units, which plays a key role in DEB modelling. First results of system dynamics are presented.

TMEM16F and dynamins control expansive plasma membrane reservoirs

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.

Photobiomodulation modulates the resolution of inflammation during acute lung injury induced by sepsis.

Sepsis is a big health problem and one of the most common causes of acute lung injury (ALI) leading to high mortality. Pro-resolving mediators play an important role in abrogating the inflammation and promoting tissue homeostasis restoration. ALI treatment is still a clinical health problem, so new therapies are needed. Here, we evaluated the effect of photobiomodulation treatment on the resolution process of ALI induced by lipopolysaccharide (LPS). Male Balb/c mice were submitted to LPS (ip) or vehicle and irradiated or not with light emitting diode (LED) 2 and 6h after LPS or vehicle injection, and the parameters were investigated 3 and 7days after the injections. Our results showed that after 3days of LED treatment the blood and bronchoalveolar lavage (BAL) cells as well as interleukins (IL) including IL-6 and IL-17 were reduced. No differences were observed in the bone marrow cells, tracheal reactivity, and lipoxin A4 and resolvin E2. Indeed, after 7days of LED treatment the bone marrow cells, lymphocytes, and lipoxin A4 were increased, while IL-6, IL-17, and IL-10 were decreased. No differences were observed in the blood cells and tracheal reactivity. Thus, our results showed that LED treatment attenuated ALI induced by sepsis by modulating the cell mobilization from their reserve compartments. In addition, we also showed later effects of the LED up to 7days after the treatment. This study proposes photobiomodulation as therapeutic adjuvant to treat ALI.

Effect of Circadian Variation on Neutrophil Mobilization to Peripheral Blood in Benign Constitutional Neutropenia

Background and Objective:Benign constitutional neutropenia (BCN) or benign ethnic neutropenia (BEN) is an asymptomatic condition characterized by mild chronic neutropenia in patients with no history of recurrent infections. In most of these patients neutropenia is discovered incidentally in a complete blood count ordered for routine screening. This is particularly concerning for general practitioners because neutropenia is associated with infections and hematologic malignancies. Thus, many patients are commonly referred to a specialist for evaluation, which leads to further testing and increased costs of care without evidence of improved personal or public health.To ensure that individuals with BCN are healthy, some diagnostic methods can be used to assess neutrophil mobilization from reserve compartments, including epinephrine, corticosteroids, endotoxins and vigorous exercise. We herein present an alternative form of assessment for individuals with BCN based on neutrophil circadian variation.Neutrophils in healthy individuals have a physiological increase of between 10 and 20% by early afternoon. In this study, we hypothesized that because patients with BCN have higher G-CSF basal levels and a greater physiological response to this cytokine, a larger increase in neutrophils can be detected in a blood test performed in the afternoon compared to routine tests performed in the morning. A physiological increase in neutrophil levels in individuals with BCN would be of great practical interest. This could reduce costs of laboratory tests and reduce medical and patient concerns.Methods:We studied patients who had neutropenia that was detected in routine blood count and with suspected BCN. Patients with clinical symptoms, laboratory test result changes and history of chronic, inflammatory, autoimmune or neoplastic diseases were excluded. Patients >60 years were excluded because they have a higher prevalence of myelodysplasia and other concomitant diseases. Neutrophil counts were evaluated in patients with BCN using paired blood counts performed in the early morning (between 7:00 and 9:00 am) and in the early afternoon (between 1:00 and 3:00 pm). The neutrophil counts were stratified into the following four groups: < 500, 500-1000, 1000-1500 and 1500-2000/µL.Results:We evaluated 157 patients with neutropenia and 118 individuals met the entry criteria. Their median age was 36 years (range, 3-60 years), and 41 (35%) were male. Median blood count results were as follows: white blood count, 2880/µL; hemoglobin, 12.9 g/dL; and platelets, 223,000/µL.The difference between the median neutrophil counts performed in the morning (1164/µL) and afternoon (2070/µL) was 904/µL (95% CI=770-1040; p<0.001). This difference reflects an increase of 94.3% in neutrophil counts between the two tests. The percent increase in neutrophils between the two assessments was higher in patients with a lower neutrophil count in the morning examination (OR=20.9; p<0.001). The lower the morning neutropenia, the greater the increase observed (513.8%, 110.6%, 70.5% and 46.8%, respectively, in the 500, 500-1000, 1000-1500 and 1500-2000/µL groups).We evaluated categories of neutrophil levels in the blood count in the morning and afternoon. Among the 118 subjects with baseline neutrophil count <2000/µL, 52 (44%) had values >2000/µL in the afternoon. Of the 65 who had remain with neutrophils <2000/µL, 48 (40%) showed an increase to neutrophil categories higher than the initial examination, 18 (15%) remained in the same category and no individual showed a decrease in neutrophil counts between the morning and afternoon.Conclusions:Our findings identify a simple, easy and feasible way to assess neutrophil reserve in patients with BCN, with a potential reduction in costs of the assessment. DisclosuresNo relevant conflicts of interest to declare.

Desvio para a esquerda associado a compartimento medular de reserva dos neutrófilos não esgotado em cadelas portadoras de piometra

From fifteen female dogs with clinical diagnoses of pyometra, hematological exams were performed at three times: M0 (prior to the surgery), M24 (24h after ovarysalpingohisterectomy-OSH) and M48 (48h after OSH). Anemia was seen in 80% of the cases, characterized as mild normocytic normochromic type. The means of total leukocyte counts were 27.043, 57.940 and 40.139 cels/µL in M0, M24 and M48. A total of80% of the animals presented neutrophilic left shift in all moments. During medullar exams, the cellular, iron reserve and megakaryocytic concentration were raised as well as the ME ratio, showing a value of 26,3:1,0, probably due to the elevation of granular proliferation and maturation compartment, as the mean of the reserve compartment was within normal range. As 83% of the animals with neutrophilic left shift showed a melullary reserve compartment raised, it can be concluded that female dogs with piometra had left shift of neutrophils with disproportionally between compartments, without segmented medullar saturation.

Inflammation induced by inactivated Aeromonas hydrophila in Nile tilapia fed diets supplemented with Saccharomyces cerevisiae

The inflammatory response and hematological parameters among Nile tilapia ( Oreochromis niloticus) supplemented with Saccharomyces cerevisiae were evaluated six and 24 h after inoculation with inactivated Aeromonas hydrophila into the swim bladder. Six groups were formed ( n = 10 each): G1 was treated with non-supplemented feed + injection with 0.65% saline solution; G2 with non-supplemented feed + inoculation with A. hydrophila; G3 with feed containing 2% yeast + injection with saline; G4 with feed containing 2% yeast + inoculation with A. hydrophila; G5 with feed containing 0.3% cell wall + injection with saline; and G6 with feed containing 0.3% cell wall + inoculation with A. hydrophila. In the groups inoculated with bacteria, the responses were more intense ( P < 0.05) than in those injected with saline. The groups receiving supplement that were inoculated with A. hydrophila accumulated a greater total number of cells at the lesion site ( P < 0.05) than did the non-supplemented groups, after six and 24 h. The groups receiving cell wall presented greater total accumulation of cells ( P < 0.005) that did those receiving yeast. The differential count showed that there were significantly greater number of thrombocytes ( P < 0.05) and lower number of neutrophils, macrophages and lymphocytes ( P < 0.05) in the groups that received supplement, after 6 and 24 h, in relation to the non-supplemented groups. The values in the erythrocyte count, hemoglobin concentration and blood measurement indices did not differ statistically. The variation in circulating thrombocyte and leukocyte counts suggests that the inflammatory stimulus caused recruitment from reserve compartments to the blood. The groups that received yeast or yeast cell wall supplements presented increased nonspecific acute inflammatory response, thus suggesting that this has a beneficial effect on the immunological defense system.

Carbohydrate storage in wood and bark of rubber trees submitted to different level of C demand induced by latex tapping

When the current level of carbohydrates produced by photosynthesis is not enough to meet the C demand for maintenance, growth or metabolism, trees use stored carbohydrates. In rubber trees (Hevea brasiliensis Muell. Arg.), however, a previous study (Silpi U., A. Lacointe, P. Kasemsap, S. Thanisawanyangkura, P. Chantuma, E. Gohet, N. Musigamart, A. Clement, T. Améglio and P. Thaler. 2007. Carbohydrate reserves as a competing sink: evidence from tapping the rubber tree. Tree Physiol. 27:881-889) showed that the additional sink created by latex tapping results not in a decrease, but in an increase in the non-structural carbohydrate (NSC) storage in trunk wood. In this study, the response of NSC storage to latex tapping was further investigated to better understand the trade-off between latex regeneration, biomass and storage. Three tapping systems were compared to the untapped Control for 2 years. Soluble sugars and starch were analyzed in bark and wood on both sides of the trunk, from 50 to 200 cm from the ground. The results confirmed over the 2 years that tapped trees stored more NSC, mainly starch, than untapped Control. Moreover, a double cut alternative tapping system, which produced a higher latex yield than conventional systems, led to even higher NSC concentrations. In all tapped trees, the increase in storage occurred together with a reduction in trunk radial growth. This was interpreted as a shift in carbon allocation toward the creation of reserves, at the expense of growth, to cover the increased risk induced by tapping (repeated wounding and loss of C in latex). Starch was lower in bark than in wood, whereas it was the contrary for soluble sugars. The resulting NSC was twice as low and less variable in bark than in wood. Although latex regeneration occurs in the bark, changes related to latex tapping were more marked in wood than in bark. From seasonal dynamics and differences between the two sides of the trunk in response to tapping, we concluded that starch in wood behaved as the long-term reserve compartment at the whole trunk level, whereas starch in bark was a local buffer. Soluble sugars behaved like an intermediate, ready-to-use compartment in both wood and bark. Finally, the dynamics of carbohydrate reserves appears a relevant parameter to assess the long-term performance of latex tapping systems.

New insights into liver stem cells

Hepatic progenitor cells are bi-potential stem cells residing in human and animal livers that are able to differentiate towards the hepatocytic and the cholangiocytic lineages. In adult livers, hepatic progenitor cells are quiescent stem cells with a low proliferating rate, representing a reserve compartment that is activated only when the mature epithelial cells of the liver are continuously damaged or inhibited in their replication, or in cases of severe cell loss. Hepatic progenitor cell activation has been described in various acute and chronic liver diseases. Their niche is composed by numerous cells such as Hepatic Stellate Cells, endothelial cells, hepatocytes, cholangiocytes, Kupffer cells, pit cells and inflammatory cells. All these cells, numerous hormones and growth factors could interact and cross-talk with progenitor cells influencing their proliferative and differentiative processes. Hepatic progenitor cells and their niche could represent, in the near future, a target for therapeutic approaches to liver disease based on cell-specific drug delivery systems. Isolation and transplantation of hepatic progenitor cells could represent a new approach for therapy of end-stage chronic liver diseases, as they offer many advantages to transplantation of mature hepatocytes. The possibility of applying stem cell therapy to liver diseases will represent a major goal in this field.

Hepatocellular stem cells

The liver has enormous regenerative capacity. Restitution of the liver in response to different injuries involves proliferation of cells at different levels of liver lineage. Mature hepatocytes, which are normally dormant, could undergo rapid replication with a near infinite capacity to proliferate. When the replication of mature hepatocytes is inhibited, a reserve compartment of bipotential hepatic progenitor/stem cells is activated. The degree of activation appears to correlate with the degree of inflammation and stage of chronic liver disease. Deregulation of key regulatory signaling pathways such as transforming growth factor-beta, Wnt, hepatocyte growth factor, insulin-like growth factor, transforming growth factor-alpha and epidermal growth factor in this progenitor/stem cell population could give rise to HCC. Further understanding of these key signaling pathways and the molecular and genetic alterations associated with HCC could provide major advances in new therapeutic and diagnostic modalities.

Telomerase Activation in Liver Regeneration and Hepatocarcinogenesis: Dr. Jekyll or Mr. Hyde?

The liver has a remarkable capability to restore its functional capacity following liver injury. According to the current paradigm, differentiated and usually quiescent hepatocytes are the primary cell type responsible for liver repair. As reserve compartment, bipotent hepatic progenitor cells are activated, especially if extensive loss or damage of hepatocytes with impaired replication occurs, e.g. in cirrhotic liver tissue. Recently, animal studies have suggested that liver regeneration following partial hepatectomy is associated with telomerase activation. Telomerase, a ribonucleoprotein with reverse transcriptase activity, plays a pivotal role in maintaining telomere length and chromosomal stability in proliferating cells. In cells lacking telomerase activity, replication-associated telomere shortening limits the replicative lifespan. Therefore, in the context of liver regeneration, telomerase activation might be a cellular mechanism to confer an extended lifespan to replicating hepatocytes and hepatic progenitor cells. On the other hand, high levels of telomerase activity are a hallmark of cancer, including hepatocellular carcinoma. Moreover, recent data indicate that telomerase activation may be an early event in hepatocarcinogenesis. At present, it is unclear, whether telomerase activation preserves the non-malignant phenotype and replicative longevity of liver cells or constitutes an early alteration obligatory for an unlimited proliferation and malignant transformation.

Inflammatory effects of BaP1 a metalloproteinase isolated from Bothrops asper snake venom: Leukocyte recruitment and release of cytokines

The inflammatory events induced by BaP1, a 22.7 kDa metalloproteinase isolated from Bothrops asper snake venom, were studied. BaP1 ip injection in mice induced a marked inflammatory cell infiltrate into peritoneal cavity of animals with predominance of neutrophils in the early phase followed by mononuclear cells in the late period. Inhibition of enzymatic activity of BaP1 by chelation with EDTA resulted in a drastic reduction of this effect. In addition, BaP1 induced a significant increase of blood neutrophil numbers before its accumulation in peritoneal cavity, thus suggesting a stimulatory action of BaP1 on mechanisms of cell mobilization from bone marrow reserve compartments. A reduction in the number of neutrophils was observed in the exudate when antibodies against LECAM-1, CD18 and LFA-1 were used, suggesting the involvement of these adhesion molecules in the effects of BaP1. In contrast, there was no effect with antibodies against ICAM-1 and PECAM-1. Moreover, a conspicuous increment in the levels of IL-1 and TNF-α, but not of LTB 4, was observed in peritoneal washes collected from mice injected with BaP1. It is concluded that BaP1 induces in vivo a marked leukocyte influx, which paralles an increased number of these cells in the blood, and is associated to the expression of specific leukocyte adhesion molecules and release of chemotactic inflammatory cytokines. Since BaP1 is a P-I class metalloproteinase, these results indicate that the proteolytic domain of metalloproteinases per se can trigger specific inflammatory events.

EcoMeristem, a model of morphogenesis and competition among sinks in rice. 2. Simulating genotype responses to phosphorus deficiency.

Phenotypic plasticity enables plants to adjust their morphology and phenology to variable environments. Although potentially important for crop breeding and management, the physiology and genetics of plasticity traits are poorly understood, and few models exist for their study. In the previous paper of this series, the structural-functional model EcoMeristem was described and field validated for vegetative-stage rice. This study applies the model to an experimental study on phosphorus deficiency effects on two morphologically contrasting rice cultivars, IR64 and Azucena, grown in controlled environments under hydroponics culture. Phosphorus deficiency caused severe biomass growth reductions in the shoot but not in the root, thus increasing the root / shoot weight ratio. It also inhibited tiller formation and leaf elongation, prolonged the phyllochron, and increased carbohydrate reserve pools in the plant. Analysis aided by the model identified inhibition of leaf extension and tillering as primary effects of the stress. Physiological feedback probably led to longer phyllochron, greater reserve accumulation and root growth stimulation. The main effect of P deficiency appeared to be a reduction in demand for assimilates in the shoot while photosynthetic radiation use efficiency remained nearly constant, resulting in spill-over of excess assimilates into reserve compartments and root growth. The results are discussed in the light of future applications of EcoMeristem for phenotyping and genetic analyses of phenotypic plasticity.

Differential regulation of rodent hepatocyte and oval cell proliferation by interferon γ†

Hepatocytes and intrahepatic progenitor cells (oval cells) have similar responses to most growth factors but rarely proliferate together. Oval cells constitute a reserve compartment that is activated when hepatocyte proliferation is inhibited. Interferon gamma (IFN-gamma) increases in liver injury that involves oval cell responses, but it is not upregulated during liver regeneration after partial hepatectomy. Based on these observations, we used well-characterized lines of hepatocytes (AML-12 cells) and oval cells (LE-6 cells) to investigate the potential mechanisms that regulate differential growth responses in hepatocytes and oval cells. We show that IFN-gamma blocks hepatocyte proliferation in vivo, and that in combination with either tumor necrosis factor (TNF) or lipopolysaccharide (LPS), it causes cell cycle arrest in hepatocytes but stimulates oval cell proliferation in cultured cells. The hepatocyte cell cycle arrest is reversible, is p53-independent, and is not associated with apoptosis. Treatment of AML-12 hepatocytes with IFN-gamma/LPS or IFN-gamma/TNF, but not with individual cytokines, induced NO synthase and generated NO, while similarly treated oval cells produced little if any NO. Generation of NO by an NO donor reproduced the inhibitory effect of the cytokine combinations on AML-12 cell replication, while NO inhibitors abolish the replication deficiency. In conclusion, we propose that IFN-gamma, in conjunction with TNF or LPS, can both inhibit hepatocyte proliferation through the generation of NO and stimulate oval cell replication. The response of hepatocytes and oval cells to cytokine combinations may contribute to the differential proliferation of these cells in hepatic growth processes.

CD34 +CDw90(Thy-1) + subset colocated with mesenchymal progenitors in human normal bone marrow hematon units is enriched in colony-forming unit megakaryocytes and long-term culture-initiating cells

Objective The progress made in the supportive care of allografts and the identification of mesenchymal stem cells in adult human bone marrow (BM) has prompted renewed interest in the use of BM as a form of cell therapy. With the aim of optimizing the collection of BM cells, we evaluated the hematopoietic and mesenchymal immature cell contents of BM hematon units (HUs), which usually are eliminated during graft processing. Materials and methods Hematopoietic CD34 + progenitors from HU and buffy coat (BC) compartments were characterized in short-term culture. The sorted CD34 +CDw90(Thy-1) + primitive subset was assessed in colony-forming cell (CFC) and long-term culture-initiating cell (LTC-IC) assays, then further characterized by the expression of additional antigens. In parallel, we evaluated the colony-forming unit fibroblast (CFU-F) number and phenotyped the fresh adherent (D1-3) cells. Results The plating efficiencies of CD34 + cells derived from HU and BC were identical. However, the HU CD34 +CDw90(Thy-1) + subset was enriched in colony-forming unit megakaryocyte (2.3×), LTC-IC (4.6×), and cells coexpressing CD105 (5×). We found a higher frequency of CFU-F (4.7×), considered to be the mesenchymal stem cell-containing population, correlated with an enrichment in fresh adherent (CD45/GPA) −CD14 − cells. Conclusions We show for the first time that functional properties of the CD34 +CDw90 + subset are related to its in vivo location in HU, which may represent the BM mesenchymal reserve compartment. The location in HU of 35.6%, 59.1%, and 58.7% of CD34 + cells, CD34 +CDw90 + LTC-IC, and CFU-F, respectively, justifies the development of a procedure to collect them in order to reduce the therapeutic BM volume.

Population genetics ofIntsia palembanica(Leguminosae) and genetic conservation of Virgin Jungle Reserves in Peninsular Malaysia

A field survey of Virgin Jungle Reserve (VJR) compartments in Peninsular Malaysia allowed us to identify six populations of Intsia palembanica for this study. These were Pasoh Forest Reserve (FR) (Pasoh), Sungai Lalang FR (Lalang), Bukit Lagong FR (Lagong), Bubu FR (Bubu), Bukit Kinta FR (Kinta), and Bukit Perangin FR (Perangin). About 40 adult individuals were sampled in each population. In addition, progeny arrays were collected from nine mother plants at Lagong for a mating system study. A total of nine allozymes, encoded by 14 putative gene loci, were consistently resolved in I. palembanica. The mating system study showed that the species exhibited a mixed-mating system, with multilocus outcrossing rate of 0.766. The levels of diversity were comparably high (mean number of alleles per polymorphic locus = 2.4, mean effective number of alleles per polymorphic locus = 1.64, and mean expected heterozygosity (H(e)) = 0.242), and the majority of the diversity was partitioned within population (G(ST) = 0.040 and F(ST) = 0.048). Significant levels of inbreeding were detected in Bubu and Perangin. Probability tests of recent effective population size reduction using the Infinite Allele Model showed the occurrence of genetic bottlenecks on Lalang and Kinta. Two genetically unique populations (Pasoh and Perangin) were inferred using jackknife analysis. By using the neutral mutation rates, effective population size (N(e)) to maintain the H(e) was 80-800 000 individuals. A simulation study based on pooled samples, however, circumscribed the N(e) to 200 and 210 individuals. Implications of the study for managing the species and the VJRs are discussed.

Modélisation de l'affinage de l'huître Crassostrea gigas alimentée avec la diatomée Skeletonema costatum

The traditional fattening of the oyster Crassostrea gigas in oyster ponds of the French Atlantic Coast is submitted to natural environmental fluctuations. In order to reduce the growth variability of the soft parts of the bivalve, an intensive fattening process was developed, where the conditions of temperature, particulate organic and inorganic matter (POM and PIM) are controlled. This process relies on the production of the diatom Skeletonema costatum, which is then distributed to the oysters at an average concentration of 4–5 mg POM·L –1. An ecophysiological model of the oyster C. gigas, which simulates the evolution of somatic and gonad–reserve compartments, was applied to these conditions in order to analyse the bivalve responses. Experimentation was performed to elaborate the model and two functions were studied: clearance rate and pseudofecal production. At a temperature of 14 °C, chosen for the fattening process, and at POM and PIM concentrations varying respectively from 4 to 18 mg·L –1 and 15 to 55 mg·L –1, it was found that clearance rate was not regulated (mean of 2.09 ± 0.11 L·h –1·g –1 with 59% of activity rate) and that ingestion rate depended on the production of pseudofeces. This production allows an increase of organic ingested fraction, by the mechanism of pre-ingestive selection that buffers the PIM variability. Negative effects of PIM on growth have been studied through several simulations. It appears that actual POM ration (4–5 mg·L –1) used in the fattening process, allows dry tissues growth, in spite of simulated PIM concentration up to 50 mg·L –1. The elaboration of the model revealed that gametogenesis is a major determinant in bivalve energy partitioning in the rich food conditions of this fattening process.

Two pathways control chromaffin cell corticalF-actin dynamics during exocytosis

Neurosecretory cells including chromaffin cells possess a mesh of filamentous actin underneath the plasma membrane. We have proposed that the F-actin network acts as a barrier to the secretory vesicles blocking their access to exocytotic sites at the plasma membrane. Disassembly of cortical F-actin in chromaffin cells in response to stimulation is thought to allow the free movement of secretory vesicles to exocytotic sites. Moreover, experiments by us using morphometric analysis of resting and stimulated chromaffin cells together with membrane capacitance measurements have shown that cortical F-actin controls the traffic of vesicles from the vesicle reserve compartment to the release-ready vesicle compartment. The dynamics of the cortical F-actin is controlled by two pathways: A) stimulation-induced Ca 2+ entry and scinderin activation; and B) protein kinase C (PKC) activation and MARCKS (myristoylated alanine-rich C kinase substrate) phosphorylation. When chromaffin cells are stimulated through nicotinic receptors, cortical F-actin disassembly is mainly through the intervention of pathway A, since in the presence of PKC inhibitors, F-actin disassembly in response to cholinergic stimulation is only blocked by 20%. Pathway A involves the activation of scinderin by Ca 2+ with a consequent F-actin severing. Pathway B is fully activated by phorbol esters and in this case PKC blockers inhibit by 100% the disruption of cortical F-actin. This pathway operates through MARCKS. A peptide with amino acid sequence corresponding to the phosphorylation site domain of MARCKS, which also corresponds to its actin binding site, blocks PMA potentiation of Ca 2+-induced catecholamine release. The results suggest that under physiological conditions (i.e., nicotinic receptor stimulation) pathway A is the principal mechanism for the control of cortical F-actin dynamic changes.

Secretory vesicle pools and rate and kinetics of single vesicle exocytosis in neurosecretory cells.

Secretory vesicles are localized in specific compartments within neurosecretory cells. Morphometric, cytochemical and electrophysiological techniques have allowed the definition of secretory vesicle compartments. These are different pools in which vesicles are in various states of releasability. The transit of vesicles between compartments is not random, but an event controlled and regulated by Ca2+ and the cortical F-actin network. Cortical F-actin disassembly, a Ca(2+)-dependent event, controls the transit of secretory vesicles from the reserve compartment to the release-ready vesicle pool. Furthermore, the recent development of new technical approaches (patch-clamp membrane capacitance, electrochemical detection of amines with carbon-fibre microelectrodes) has now permitted us to understand the kinetics of single vesicle exocytosis.

Reactive biliary epithelium: The product of a pluripotential stem cell compartment?

Liver parenchymal cells (hepatocytes) have a low rate of turn-over, but can nevertheless mount a rapid and efficient regenerative response. However, in some cases of extreme hepatotoxicity hepatocyte proliferation is restricted or even abolished, and instead biliary epithelial cells, commonly referred to as ductular oval cells, migrate into the periportal and midzonal parenchyma. Initially these cells behave as authentic biliary epithelium with expression of the biliary cytokeratin intermediate filaments, but then show hepatocytic traits such as alpha fetoprotein and albumin synthesis. Thereafter these biliary ducts rapidly vanish to be replaced by either small hepatocytes or intestinal-type cells. The proliferation and differentiation of oval cells is probably strongly influenced by paracrine signalling from liver stellate cells. Oval cells appear to be the progeny of facultative pluri-potential stem cells which have the lineage potential of uncommitted gastrointestinal stem cells; these stem cells are likely to be located in the cholangioles and small interlobular bile ducts. Oval cells thus constitute an important reserve compartment for hepatocytes when hepatocyte regeneration is compromised.

Capillary electrophoresis of single cells: observation of two compartments of neurotransmitter vesicles

Capillary electrophoresis has been used to directly identify and measure the neurotransmitter, dopamine, in two vesicular compartments in a single nerve cell of Planorbis corneus. Dopamine in the cytoplasm and in easily released transmitter vesicles was separated from dopamine in what are apparently non-functional storage vesicles. In this method, the two peaks in the electropherogram attributed to dopamine were differentiated based on cell lyse time in a non-physiological buffer. The measurements presented here suggest that of the total dopamine present in the cell: 24% is in the cytoplasmic and easily released compartments and 76% is more centrally located, perhaps in a reserve compartment, in the cell. This methodology provides the means to determine molecular species in subcellular compartments and should allow kinetic parameters associated with membrane lysing to be evaluated at single nerve cells.