Cytoplasmic Fragments Research Articles

Biological Variation of the Platelet Proteome in the Elderly Population and Its Implication for Biomarker Research

Knowledge about the extent of total variation experienced between samples from different individuals is of great importance for the design of not only proteomics but every clinical study. This variation defines the smallest statistically significant detectable signal difference when comparing two groups of individuals. We isolated platelets from 20 healthy human volunteers aged 56-100 years because this age group is most commonly encountered in the clinics. We determined the technical and total variation experienced in a proteome analysis using two-dimensional DIGE with IPGs in the pI ranges 4-7 and 6-9. Only spots that were reproducibly detectable in at least 90% of all gels (n = 908) were included in the study. All spots had a similar technical variation with a median coefficient of variation (cv) of about 7%. In contrast, spots showed a more diverse total variation between individuals with a surprisingly low median cv of only 18%. Because most known biomarkers show an effect size in a 1-2-fold range of their cv, any future clinical proteomics study with platelets will require an analytical method that is able to detect such small quantitative differences. In addition, we calculated the minimal number of samples (sample size) needed to detect given protein expression differences with statistical significance.

Chromosomes are eliminated in the symmetric fusion between Arabidopsis thaliana L. and Bupleurum scorzonerifolium Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed.

Abstract 2677: Reduction In Tie-2+/flk-1+ Vascular Stem Cells In Thoracic Aortic Aneurysm With Dissections: A Novel Contributor To Acellular Lesion Development

Background. Ascending thoracic aortic aneurysms leading to type A dissections (TAAD) are a life-threatening disease triggered by a complex interactions between environmental and genetic factors. Pathologically, this disease is characterized by a progressive loss of vascular smooth muscle cells (SMCs) and extracellular elastic fibers in the aortic media. Apoptosis, a form of programmed cell death, has been shown to contribute to the loss of medial SMCs. However, little is known about the role of vascular progenitor or stem cells in the pathogenesis of aortic aneurysms. Methods and Results. In this study we examined expression of Tie-2 (receptor for angiopoietin) and Flk-1 (receptor for vascular endothelial cell growth factor), two receptors critical for regulation of vascular stem cell survival and growth in the aortic lesions of patients with TAAD. Immunohistochemistry with specific antibodies against Tie-2 and Flk-1 showed a marked reduction in the numbers of Tie+/Flk+ cells in TAAD when compared to control aortas. There was also a change in the distribution pattern of vascular progenitor cells positive for both Tie-2 and Flk-1 in TAAD patients’ aortic tissues. Compared to normal control aortas, the Tie-2 and Flk-1 immunostained cells were much lower in intensity and scattered. In the regions with paucity of Tie-2+/Flk-1+ cells, there was an increased number of apoptotic cells as determined by in situ DNA end-labeling (TUNEL). Many of the Tie-2 and Flk-1 positive cells in TAAD showed poor morphology with nuclear degeneration and cytoplasmic fragmentation and disarray. Conclusion . These results indicate a paucity of Tie-2+/Flk-1+ vascular stem cells in the aortic wall of patients with TAAD, implying that the stem cell capacity to regenerate SMCs is exhausted. Similar loss of stem cells is observed with Duchenne muscular dystrophy, where the regenerated capacity is significantly compromised. Stem cells loss may similarly impair the capacity of SMCs in aortic tissue in repair and regeneration, and ultimately lead to TAAD formation.

Vascular Endothelial Growth Factor and Nitric Oxide Production in Response to Hypoxia in the Choroid Plexus in Neonatal Brain

Damage to the choroid plexus in 1-day-old Wistar rats subjected to hypoxia was investigated. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal, inducible nitric oxide synthase (eNOS, nNOS, iNOS), and vascular endothelial growth factor (VEGF) along with nitric oxide (NO) production and VEGF concentration was up-regulated significantly in hypoxic rats. Ultrastructurally, the choroid plexus epithelial cells showed massive accumulation of glycogen. A striking feature was the extrusion of cytoplasmic fragments from the apical cell surfaces into the ventricular lumen following the hypoxic insult. Intraventricular macrophages showed increased expression of complement type 3 receptors, major histocompatibility complex class I and II antigens, and ED1 antigens. Following an intravenous injection of horseradish peroxidase (HRP), a large number of intraventricular macrophages were labeled suggesting enhanced leakage of the tracer from the blood vessels in the choroid plexus connective tissue stroma into the ventricular lumen. We suggest that increased production of NO in hypoxia is linked to the structural alteration of the choroid plexus, and along with VEGF, may lead to increased vascular permeability. Melatonin treatment reduced VEGF and NO levels as well as leakage of HRP suggesting its potential value in ameliorating damage in choroid plexus pathologies.

L-carnitine as an antiapoptotic supplement in mouse embryo culture media

ObjectiveHuman embryos generated from in vitro fertilization (IVF) show varying degrees of cytoplasmic fragmentation. Reports suggest that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. L-Carnitine (LC) has no major side effects on embryonic cells, interactions with other drugs or teratogenicity. It acts by stabilizing the mitochondrial membrane and protecting the cell from apoptosis. The aim of our study was to investigate the antiapoptotic effect of LC supplementation in mouse embryo culture media.DesignProspective in vitro study.Materials and methodsA total of 240 mouse embryos (8-cell) were randomly assigned into 4 groups: group 1: control (HTF media only); groups 2 (Actinomycin D 5 ng/mL; dose based on previous literature) group 3–4: (Actinomycin D 5 ng/mL + two concentrations of LC (0.3 and 0.6 mg/mL). LC concentrations were based on pilot study by our group to optimize the dose of LC in mouse embryo culture media. Embryos were incubated at 37°C in 5% CO2. Assessment of embryos development was done after 48 hours by examining the percentage blastocyst development rate (%BDR). After TUNEL staining, the blastocysts were examined by confocal microscopy. Apoptosis was measured by examining the percentage of TUNEL positive blastomeres.ResultsTableaP<0.05 considered significant using Fisher's exact test for %BDR differences; bP<0.05 was significant for differences in apoptosis using Kruskal-Wallis or Wilcoxon test. cP differences between Actinomycin group and Actinomycin + LC groups. Open table in a new tab ConclusionsL-Carnitine improves the blastocyst development rate and reduces apoptosis. Supplementing in vitro culture media with L-Carnitine may improve the fertilization outcome. ObjectiveHuman embryos generated from in vitro fertilization (IVF) show varying degrees of cytoplasmic fragmentation. Reports suggest that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. L-Carnitine (LC) has no major side effects on embryonic cells, interactions with other drugs or teratogenicity. It acts by stabilizing the mitochondrial membrane and protecting the cell from apoptosis. The aim of our study was to investigate the antiapoptotic effect of LC supplementation in mouse embryo culture media. Human embryos generated from in vitro fertilization (IVF) show varying degrees of cytoplasmic fragmentation. Reports suggest that cytoplasmic fragmentation in human embryos arises from apoptosis. Mitochondrial membrane potential and energy production in preimplantation embryos have recently been the focus of many studies. L-Carnitine (LC) has no major side effects on embryonic cells, interactions with other drugs or teratogenicity. It acts by stabilizing the mitochondrial membrane and protecting the cell from apoptosis. The aim of our study was to investigate the antiapoptotic effect of LC supplementation in mouse embryo culture media. DesignProspective in vitro study. Prospective in vitro study. Materials and methodsA total of 240 mouse embryos (8-cell) were randomly assigned into 4 groups: group 1: control (HTF media only); groups 2 (Actinomycin D 5 ng/mL; dose based on previous literature) group 3–4: (Actinomycin D 5 ng/mL + two concentrations of LC (0.3 and 0.6 mg/mL). LC concentrations were based on pilot study by our group to optimize the dose of LC in mouse embryo culture media. Embryos were incubated at 37°C in 5% CO2. Assessment of embryos development was done after 48 hours by examining the percentage blastocyst development rate (%BDR). After TUNEL staining, the blastocysts were examined by confocal microscopy. Apoptosis was measured by examining the percentage of TUNEL positive blastomeres. A total of 240 mouse embryos (8-cell) were randomly assigned into 4 groups: group 1: control (HTF media only); groups 2 (Actinomycin D 5 ng/mL; dose based on previous literature) group 3–4: (Actinomycin D 5 ng/mL + two concentrations of LC (0.3 and 0.6 mg/mL). LC concentrations were based on pilot study by our group to optimize the dose of LC in mouse embryo culture media. Embryos were incubated at 37°C in 5% CO2. Assessment of embryos development was done after 48 hours by examining the percentage blastocyst development rate (%BDR). After TUNEL staining, the blastocysts were examined by confocal microscopy. Apoptosis was measured by examining the percentage of TUNEL positive blastomeres. ResultsTableaP<0.05 considered significant using Fisher's exact test for %BDR differences; bP<0.05 was significant for differences in apoptosis using Kruskal-Wallis or Wilcoxon test. cP differences between Actinomycin group and Actinomycin + LC groups. Open table in a new tab aP<0.05 considered significant using Fisher's exact test for %BDR differences; bP<0.05 was significant for differences in apoptosis using Kruskal-Wallis or Wilcoxon test. cP differences between Actinomycin group and Actinomycin + LC groups. ConclusionsL-Carnitine improves the blastocyst development rate and reduces apoptosis. Supplementing in vitro culture media with L-Carnitine may improve the fertilization outcome. L-Carnitine improves the blastocyst development rate and reduces apoptosis. Supplementing in vitro culture media with L-Carnitine may improve the fertilization outcome.

L-carnitine improves blastocyst development rate and reduces DNA damage in mouse embryos

Cytoplasmic fragmentation may be present in the in-vitro cultured embryos. This fragmentation may arise from apoptosis. Apoptosis is associated with changes in the mitochondrial membrane potential. Recent studies show that ooplasmic injection of isolated mitochondria can rescue oocyte fragmentation in mice. L-Carnitine (LC) is able to stabilize the mitochondrial membranes potential, increase the supply of energy and protect the cell from apoptotic death. In a pilot study we have shown that L Carnitine can improve the blastocysts development rate (%BDR) in 2-cell embryos at low concentrations (0.3 mg/ml).

TAZ Promotes PC2 Degradation through a SCFβ-Trcp E3 Ligase Complex

Studies of a TAZ knockout mouse reveal a novel function of the transcriptional regulator TAZ, that is, as a binding partner of the F-box protein beta-Trcp. TAZ-/- mice develop polycystic kidney disease (PKD) and emphysema. The calcium-permeable cation channel protein polycystin 2 (PC2) is overexpressed in kidneys of TAZ-/- mice as a result of decreased degradation via an SCF(beta-Trcp) E3 ubiquitin ligase pathway. Replacements of serines in a phosphodegron motif in TAZ prevent beta-Trcp binding and PC2 degradation. Coexpression of a cytoplasmic fragment of polycystin 1 blocks the PC2-TAZ interaction and prevents TAZ-mediated degradation of PC2. Depletion of TAZ in zebrafish also results in a cystic kidney accompanied by overexpression of PC2. These results establish a common role of TAZ across vertebrate species in a protein degradation pathway regulated by phosphorylation and implicate deficiencies in this pathway in the development of PKD.

Megakaryocytes and platelet homeostasis in diffuse alveolar damage

Platelet homeostasis reflects a balance between the production of platelets via cytoplasmic fragmentation of megakaryocytes in the pulmonary microvasculature and their catabolism. Increased numbers of megakaryocytes are entrapped in the injured lung, potentially affecting circulating platelet counts. We enumerated pulmonary megakaryocytes and blood platelets in patients with diffuse alveolar damage (DAD) in order to determine their association with clinical outcome. Lung biopsies were examined from 21 patients with histologically documented DAD in its proliferative phase and secondary to a variety of causes. Blood platelet counts were determined within 24 h prior to lung biopsy, and CD61+ pulmonary megakaryocytes were localized in in situ immunohistochemical stains. The overall mortality in this series was 67%. Patients with DAD attributable to drug toxicity (DAD-D) had higher mortality (80%) and greater number of intrapulmonary CD61+ megakaryocytes than those with DAD due to other causes (23 ± 7, 10 ± 2, p < 0.05). Patients with blood platelet counts = 350 th/cm 3 showed increased survival ( p < 0.05). The findings support the hypothesis that abnormal platelet homeostasis is associated with increased mortality in acute lung injury and indicate that thrombocytosis in ARDS is associated with improved survival. The mechanisms of altered platelet homeostasis in DAD merit further investigation.

Rho-kinase inhibitor prevents hepatocyte damage in acute liver injury induced by carbon tetrachloride in rats

A protective effect of Rho-kinase inhibitor on various organ injuries is gaining attention. Regarding liver injury, Rho-kinase inhibitor is reported to prevent carbon tetrachloride (CCl4)- or dimethylnitrosamine-induced liver fibrosis and hepatic ischemia-reperfusion injury in rats. Because Rho-kinase inhibitor not only improved liver fibrosis but also reduced serum alanine aminotransferase (ALT) level in CCl4-induced liver fibrosis, we wondered whether Rho-kinase inhibitor might exert a direct hepatocyte-protective effect. We examined this possibility in acute CCl4 intoxication in rats. Rho-kinase inhibitor, HA-1077, reduced serum alanine ALT level in rats with acute liver injury induced by CCl4 with the improvement of histological damage and the reduction of the number of apoptotic cells. In cultured rat hepatocytes in serum-free condition, HA-1077 reduced apoptosis evaluated by quantitative determination of cytoplasmic histone-associated DNA oligonucleosome fragments with the reduction of caspase-3 activity and the enhancement of Bcl-2 expression. HA-1077 stimulated phosphorylation of Akt, and wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway, abrogated the reduction of hepatocyte apoptosis by HA-1077 in vitro. Furthermore, wortmannin abrogated the reduction of serum ALT level by HA-1077 in rats with acute liver injury induced by CCl4, suggesting that the activation of PI3-kinase/Akt pathway may be involved in the hepatocyte-protective effect by Rho-kinase inhibitor in vivo. In conclusion, Rho-kinase inhibitor prevented hepatocyte damage in acute liver injury induced by CCl4 in rats and merits consideration as a hepatocyte-protective agent in liver injury, considering its direct antiapoptotic effect on hepatocytes in vitro.

Endoplasmic Reticulum-Associated Degradation of Growth Hormone Receptor in Janus Kinase 2-Deficient Cells

A key factor governing cellular sensitivity to GH is cell surface GH receptor (GHR) abundance, which is affected transcriptionally and posttranscriptionally. Mature cell surface GHR abundance is regulated by constitutive and inducible metalloproteolysis and constitutive endosomal/lysosomal degradation. We previously found that Janus kinase 2 (JAK2)-deficient GHR-expressing cells have a greater precursor/mature GHR ratio, exhibit diminished inducible metalloproteolysis, and have a cytoplasmic domain-containing GHR fragment called the basal remnant (by virtue of comigration on SDS-PAGE with the inducible, metalloprotease-generated remnant). Herein we examined the mechanism of generation of basal remnant in JAK2-deficient cells, asking whether it originates from precursor vs. mature receptor and which protease(s) catalyzes its appearance. Prolonged metalloprotease inhibitor treatment or small interfering RNA knockdown of TNF-alpha converting enzyme (TACE) and a disintegrin and metalloprotease-10 (ADAM10) (both implicated in inducible GHR proteolysis) did not reduce basal remnant, indicating its generation is not metalloprotease dependent. However, a mutant GHR resistant to metalloprotease cleavage did not yield basal remnant when expressed in JAK2-deficient cells, suggesting common structural determinants for generation of the inducible remnant and the non-metalloprotease-generated basal remnant seen in JAK2-deficient cells. Treatment of JAK2-deficient cells with a proteasome inhibitor, but not two separate lysosome inhibitors, dramatically decreased basal remnant, accompanied by decreased precursor GHR and increased mature GHR abundance. Disruption of endoplasmic reticulum-to-Golgi transport with brefeldin A (BFA) also reduced basal remnant, and washout of BFA allowed regeneration of basal remnant along with GHR precursor. Notably, BFA washout in the presence of cycloheximide blocked both basal remnant and precursor GHR reappearance, but BFA washout in the presence of lactacystin blocked only basal remnant reappearance, suggesting that basal remnant is generated proteasome dependently from precursor GHR. Collectively, our data suggest that JAK2, by association with GHR in the secretory pathway, blunts proteasome activity-dependent discrete GHR cleavage and endoplasmic reticulum-dependent degradation of the precursor receptor. In so doing, JAK2 enables efficient processing of precursor receptor to mature GHR.

ErbB4 Isoforms Selectively Regulate Growth Factor–induced Madin-Darby Canine Kidney Cell Tubulogenesis

ErbB4, a member of the epidermal growth factor (EGF) receptor family that can be activated by heregulin beta1 and heparin binding (HB)-EGF, is expressed as alternatively spliced isoforms characterized by variant extracellular juxtamembrane (JM) and intracellular cytoplasmic (CYT) domains. ErbB4 plays a critical role in cardiac and neural development. We demonstrated that ErbB4 is expressed in the ureteric buds and developing tubules of embryonic rat kidney and in collecting ducts in adult. The predominant isoforms expressed in kidney are JM-a and CYT-2. In ErbB4-transfected MDCK II cells, basal cell proliferation and hepatocyte growth factor (HGF)-induced tubule formation were decreased by all four isoforms. Only JM-a/CYT-2 cells formed tubules upon HB-EGF stimulation. ErbB4 was activated by both HRG-beta1 and HB-EGF stimulation; however, compared with HRG-beta1, HB-EGF induced phosphorylation of the 80-kDa cytoplasmic cleavage fragment of the JM-a/CYT-2 isoform. HB-EGF also induced early activation of ERK1/2 in JM-a/CYT-2 cells and promoted nuclear translocation of the JM-a/CYT-2 cytoplasmic tail. In summary, our data indicate that JM-a/CYT-2, the ErbB4 isoform that is proteinase cleavable but does not contain a PI3K-binding domain in its cytoplasmic tail, mediates important functions in renal epithelial cells in response to HB-EGF.

Insulysin Cleaves the APP Cytoplasmic Fragment at Multiple Sites

The amyloid peptide (Abeta) deposited in Alzheimer's disease (AD) is generated by beta- and gamma-secretase processing of a larger integral membrane protein precursor (APP). Intramembrane processing of APP by gamma-secretase also yields an intracellular fragment, CTFgamma (a.k.a. AICD), which is highly conserved and is believed to regulate the transcription of several genes including KAI-1 and GSK3beta. The intracellular domain of APP is also processed by caspase to a 31 aa fragment that was shown to induce apoptosis by several groups. Although large quantities of CTFgamma are generated continuously by neurons, little if any is normally detected in cell lysates, which suggests that it is very rapidly turned over in vivo. Previous studies demonstrated that insulysin (IDE), an Abeta-degrading enzyme, is responsible for cytosol-mediated CTFgamma degradation in vitro. Consistent with this finding, knockout mice lacking IDE accumulate CTFgamma to detectable levels in the brain, although its levels remain lower than its precursor, suggesting that it continues to be turned over in the brain. Moreover, when we treated cultured cells with IDE inhibitors, we did not observe an increase in CTFgamma in cell lysates, suggesting that pathways other than IDE are also involved in CTFgamma turnover. To understand CTFgamma turnover further, we have mapped the IDE cleavage sites with the intention of mutating them to examine alternative pathways in future studies. Edman degradation revealed that IDE cleaves CTFgamma at multiple sites to small peptides ranging from 5 to 14 aa. The cleavage sites do not reveal the existence of any sequence specificity for IDE cleavage. Understanding the turnover mechanisms of CTFgamma is critical to the understanding of the signaling function of APP mediated by this fragment. The current study presents the interesting specificity of CTFgamma turnover by IDE, which has been previously identified as the major degrading enzyme for Abeta as well as CTFgamma. In addition, the study provides evidence for the presence of alternative CTFgamma-degrading pathways in the cell.

Phylogeny and Biogeography of Cedrus (Pinaceae) Inferred from Sequences of Seven Paternal Chloroplast and Maternal Mitochondrial DNA Regions

Cedrus (true cedars) is a very important horticultural plant group. It has a disjunct distribution in the Mediterranean region and western Himalaya. Its evolution and biogeography are of great interest to botanists. This study aims to investigate the phylogeny and biogeography of Cedrus based on sequence analyses of seven cytoplasmic DNA fragments. The methods used were PCR amplification and sequencing of seven paternal cpDNA and maternal mtDNA fragments, parsimony and maximum likelihood analyses of the DNA dataset, and molecular clock estimate of divergence times of Cedrus species. Phylogenies of Cedrus constructed from cpDNA, mtDNA and the combined cp- and mt-DNA dataset are identical in topology. It was found that the Himalayan cedar C. deodara diverged first, and then the North African species C. atlantica separated from the common ancestor of C. libani and C. brevifolia, two species from the eastern Mediterranean area. Molecular clock estimates suggest that the divergence between C. atlantica and the eastern Mediterranean clade at 23.49 +/- 3.55 to 18.81 +/- 1.25 Myr and the split between C. libani and C. brevifolia at 7.83 +/- 2.79 to 6.56 +/- 1.20 Myr. The results, combined with palaeogeographical and palaeoecological information, indicate that Cedrus could have an origin in the high latitude area of Eurasia, and its present distribution might result from vicariance of southerly migrated populations during climatic oscillations in the Tertiary and further fragmentation and dispersal of these populations. It is very likely that Cedrus migrated into North Africa in the very late Tertiary, while its arrival in the Himalayas would not have been before the Miocene, after which the phased or fast uplift of the Tibetan plateau happened.

The cytosolic N-terminus of presenilin-1 potentiates mouse ryanodine receptor single channel activity

Ryanodine receptors (RyRs) amplify intracellular Ca 2+ signals by massively releasing Ca 2+ from intracellular stores. Exaggerated chronic Ca 2+ release can trigger cellular apoptosis underlying a variety of neurodegenerative diseases. Aberrant functioning of presenilin-1 (PS1) protein instigates Ca 2+-dependent apoptosis, providing a basis for the “calcium hypothesis” of Alzheimer's disease (AD). To get insight into this problem, we hypothesized that the previously reported physical interaction between RyR and PS1 modulates functional properties of the RyR. We generated a soluble cytoplasmic N-terminal fragment of PS1 comprising the first 82 amino acid (PS1 NTF 1–82), the candidate for interaction with putative cytoplasmic modulatory sites of the RyR, and studied its effect on single channel currents of mouse brain RyRs incorporated in lipid bilayers. PS1 NTF 1–82 strongly increased both mean currents (EC 50 = 12 nM, Hill coefficient ( n H) ∼ 1) and open probability for higher sublevels for single RyR channels (EC 50 = 7 nM, n H ∼ 2). Bell-shaped Ca 2+-activation curve remained unchanged, suggesting that PS1 NTF 1–82 allosterically potentiates RyRs, but that the channel still requires Ca 2+ for activation. Corroborating such an independent mechanism, the RyR potentiation by PS1 NTF 1–82 was overridden by receptor desensitization at high [Ca 2+] ( pCa > 5). This potentiation of RyR by PS1 NTF 1–82 reveals a new mechanism of physiologically relevant PS1-regulated Ca 2+ release from intracellular stores, which could be alternative or additional to recently reported intracellular Ca 2+ leak channels formed by PS1 holoproteins.

Characterisation of cell death in bagged baby salad leaves

Baby leaf salads are gaining in popularity over traditional whole head lettuce salads in response to consumer demand for greater variety and convenience in their diet. Baby lettuce leaves are mixed, washed and packaged as whole leaves, with a shelf-life of approximately 10 days post-processing. End of shelf-life, as determined by the consumer, is typified by bruising, water-logging and blackening of the leaves, but the biological events causing this phenotype have not been studied to date. We investigated the physiological and ultrastructural characteristics during postharvest shelf-life of two lettuce varieties with very different leaf morphologies. Membrane disruption was an important determinant of cell death in both varieties, although the timing and characteristics of breakdown was different in each with Lollo rossa showing signs of aging such as thylakoid disruption and plastoglobuli accumulation earlier than Cos. Membranes in Lollo rossa showed a later, but more distinct increase in permeability than in Cos, as indicated by electrolyte leakage and the presence of cytoplasmic fragments in the vacuole, but Cos membranes show distinct fractures towards the end of shelf-life. The tissue lost less than 25% fresh weight during shelf-life and there was little protein loss compared to developmentally aging leaves in an ambient environment. Biophysical measurements showed that breakstrength was significantly reduced in Lollo rossa, whereas irreversible leaf plasticity was significantly reduced in Cos leaves. The reversible elastic properties of both varieties changed throughout shelf-life. We compared the characteristics of shelf-life in both varieties of bagged lettuce leaves with other leafy salad crops and discuss the potential targets for future work to improve postharvest quality of baby leaf lettuce.

From hematopoietic stem cells to platelets.

Megakaryocytopoiesis is the process that leads to the production of platelets. This process involves the commitment of multipotent hematopoietic stem cells toward megakaryocyte (MK) progenitors, the proliferation and differentiation of MK progenitors, the polyploidization of MK precursors and the maturation of MK. Mature MK produce platelets by cytoplasmic fragmentation occurring through a dynamic and regulated process, called proplatelet formation, and consisting of long pseudopodial elongations that break in the blood flow. Recent insights have demonstrated that the MK and erythroid lineages are tightly associated at both the cellular and molecular levels, especially in the transcription factors that regulate their differentiation programs. Megakaryocytopoiesis is regulated by two types of transcription factors, those regulating the differentiation process, such as GATA-1, and those regulating proplatelet formation, such as NF-E2. The humoral factor thrombopoietin (TPO) is the primary regulator of MK differentiation and platelet production through the stimulation of its receptor MPL. Numerous acquired or congenital pathologies of the MK lineage are now explained by molecular abnormalities in the activity of the transcription factors involved in megakaryocytopoiesis, in the Tpo or c-mpl genes, as well as in signaling molecules associated with MPL. The recent development of MPL agonists may provide efficient agents for the treatment of some thrombocytopenias.

T11TS/SLFA-3 Differentially Regulate the Population of Microglia and Brain Infiltrating Lymphocytes to Reduce Glioma by Modulating Intrinsic Bcl-2 Expression rather than p53

T11TS/SLFA-3, the glycoprotein isolated from sheep erythrocyte membrane, acts as an antineoplastic agent causing apoptotic elimination of glioma cells through cell mediated immune response. Therefore, elucidation of the proper balance in proliferation and apoptosis of neuroimmune components viz. microglia and brain infiltrating lymphocytes with the neoplastic glial cells was the fundamental issue to establish the efficacy of T11TS as a therapeutic agent in glioma. To decipher its effectivity on proliferation rate of glioma cells, expression of GFAP and cell cycle phase distribution was analyzed with propidium iodide (PI) staining. The apoptotic regulation of interacting immuno-competent microglia, lymphocytes entering into the brain and target glioma cells were elucidated by cytoplasmic DNA fragmentation assay and phosphatidylserine (PS) externalization along with intrinsic p53 and Bcl-2 modulation of the cells. With the reduction of cellular proliferation rate, sharp increase of apoptosis in consecutive doses of T11TS showed the regression of glioma, where an increase of cytosolic p53 and a decrease of Bcl-2 with doses in these neoplastic cells facilitate the process. Resident microglia, the chief immunomodulator of brain, was found to show a low and steady level of proliferation and apoptosis, furnishing it as a stable pool of cells capable of controlling immune reaction in brain compartment. However, microglia showed higher basal level of p53 compared to the other cells in study and being modulated with T11TS dose, it was found to possess a steady level of Bcl-2 that aided to maintain low rate of apoptosis. Brain infiltrating lymphocytes showed increased apoptosis in tumorigenic condition and initial treatment phase mostly due to immuno-suppressive milieu and deprivation of microglial restimulation. But the second dose of T11TS showed reduced apoptosis, enhanced activation of lymphocytes and final dose acting as a regulatory dose, which reduce the infiltrating lymphocytes by apoptotic elimination. Wide fluctuation of cytosolic p53 was observed in these lymphocytes, but anti-apoptotic Bcl-2 was found to modulate apoptosis in the cells. Thus, T11TS was found to differentially regulate the population of immune effector cells against glioma to exert effective effector function in eradication of neoplastic cells where Bcl-2 constitutively suppresses pro-apoptotic function of p53. Regulation of the direction of these balances of cellular life and death in favor of glioma killing and also maintaining homeostasis in brain tissue after reaction established T11TS as an effective therapeutic probe against glioma.

Development to the Blastocyst Stage of Porcine Somatic Cell Nuclear Transfer Embryos Reconstructed by the Fusion of Cumulus Cells and Cytoplasts Prepared by Gradient Centrifugation

The present study was designated to examine the possibility of producing somatic cell nuclear transfer (SCNT) embryos in pigs using oocyte cytoplasm fragments (OCFs), prepared by centrifugations, as recipient cytoplasts. In Experiment 1, in vitro matured oocytes were centrifuged at 13,000 x g for 3, 6, and 9 min to stratify the cytoplasm, and then the oocytes were freed from zona pellucida and recentrifuged at 5,000 x g for 4 sec in Percoll gradient solution to produce OCFs as the source of recipient cytoplasts. It was found that a long duration of the first centrifugation tends to produce large-sized OCFs after the second centrifugation. In Experiment 2, two or three cytoplasts without chromosomes were aggregated, and then they were fused with a cumulus cell to produce SCNT embryos. The results showed that 66.4 +/- 9.4% of the reconstructed embryos underwent premature chromosome condensation at 1 h after activation, and 85.2 +/- 7.1% and 61.6 +/- 7.0% of them had pseudopronuclei at 10 and 24 h after activation, respectively. In Experiment 3, when SCNT embryos reconstructed by the fusion of three cytoplasts and one cumulus cell, a significantly higher (p < 0.05) rate of reconstructed embryos developed to the blastocyst stage (10.6 +/- 1.8%) than that of reconstructed with two cytoplasts and one cumulus cell (5.2 +/- 1.5%). These results indicate that cytoplasts obtained by two centrifugations can support the remodeling of a transferred somatic nucleus, resulting in the development of the reconstructed porcine embryos to the blastocyst stage.

Ectodomain shedding of neuroglycan C, a brain‐specific chondroitin sulfate proteoglycan, by TIMP‐2‐ and TIMP‐3‐sensitive proteolysis

Neuroglycan C (NGC) is a transmembrane-type of chondroitin sulfate proteoglycan with an epidermal growth factor (EGF)-like module that is exclusively expressed in the CNS. Because ectodomain shedding is a common processing step for many transmembrane proteins, we examined whether NGC was subjected to proteolytic cleavage. Western blotting demonstrated the occurrence of a soluble form of NGC with a 75 kDa core glycoprotein in the soluble fraction of the young rat cerebrum. In contrast, full-length NGC with a 120 kDa core glycoprotein and its cytoplasmic fragment with a molecular size of 35 kDa could be detected in the membrane fraction. The soluble form of NGC was also detectable in culture media of fetal rat neurons, and the full-length form existed in cell layers. The amount of the soluble form in culture media was decreased by adding a physiological protease inhibitor such as a tissue inhibitor of metalloproteinase (TIMP)-2 or TIMP-3, but not by adding TIMP-1. Both EGF-like and neurite outgrowth-promoting activity of the NGC ectodomain may be regulated by this proteolytic processing.

Intracellular renin-angiotensin system: the tip of the intracrine physiology iceberg

it is axiomatic that peptide hormones bind to cognate receptors on target cells, with the resultant generation of intracellular second messengers. Not as well known is the fact that a large and growing body of evidence indicates that extracellular signaling peptides, such as hormones and growth