CONGENITAL ANOMALIES, Issue 4 2008
Mikio Nakajima
ABSTRACT Indium, a precious metal classified in group 13 (IIIB) in the periodic table, has been used increasingly in the semiconductor industry. Because indium is a rare metal, technology for indium recycling from transparent conducting films for liquid crystal displays is desired, and its safety evaluation is becoming increasingly necessary. The developmental toxicity of indium in experimental animals was summarized. The intravenous or oral administration of indium to pregnant animals causes growth inhibition and the death of embryos in hamsters, rats, and mice. The intravenous administration of indium to pregnant animals causes embryonic or fetal malformation, mainly involving digit and tail deformities, in hamsters and rats. The oral administration of indium also induces fetal malformation in rats and rabbits, but requires higher doses. No teratogenicity has been observed in mice. Caudal hypoplasia, probably due to excessive cell loss by increased apoptosis in the tailbud, in the early postimplantation stage was considered to account for indium-induced tail malformation as a possible pathogenetic mechanism. Findings from in vitro experiments indicated that the embryotoxicity of indium could have direct effects on the conceptuses. Toxicokinetic studies showed that the embryonic exposure concentration was more critical than the exposure time regarding the embryotoxicity of indium. It is considered from these findings that the risk of the developmental toxicity of indium in humans is low, unless an accidentally high level of exposure or unknown toxic interaction occurs because of possible human exposure routes and levels (i.e. oral, very low-level exposure). [source]

Cell death in normal and abnormal development

CONGENITAL ANOMALIES, Issue 1 2008
Philip E. Mirkes
ABSTRACT Research over the past 50 years has consistently documented that cell death is an integral part of both normal development and the etiology of birth defects; however, the significance of this cell death has been, until recently, unclear. Research published during the past 15 years has now shown that programmed cell death (PCD) and teratogen-induced cell death are genetically controlled processes (apoptosis) that play important roles in both normal and abnormal development. Therefore, the purpose of this review is to highlight what is known about PCD and teratogen-induced cell death and their relationships to the mechanisms of apoptosis and abnormal development. [source]

Thalidomide for the treatment of multiple myeloma

CONGENITAL ANOMALIES, Issue 3 2004
Yutaka Hattori
ABSTRACT Although thalidomide was withdrawn in the 1960s after its teratogenic property was recognized, it was subsequently found that this drug possesses immunomodulatory and anti-inflammatory effects. Recent studies have also demonstrated that thalidomide has antineoplastic activity via an antiangiogenic mechanism. Observations in the late 1990s that the microenvironment in the bone marrow plays a role in tumor progression in multiple myeloma provided an impetus to use thalidomide for the treatment of this disease. It is known that thalidomide monotherapy is effective in one-third of refractory cases, and in combination with glucocorticoids and/or antineoplastic drugs, thalidomide provides a response rate of more than 50%. Thus, thalidomide therapy is considered a standard approach for the treatment of relapsed and refractory myeloma. The exact mechanism of the antimyeloma effect of thalidomide is not yet clearly understood. Anti-angiogenic effects, direct activity in tumor cells such as the induction of apoptosis or G1 arrest of the cell cycle, the inhibition of growth factor production, the regulation of interactions between tumor and stromal cells, and the modulation of tumor immunity have been considered as possible mechanisms. In addition to its teratogenicity, the adverse effects of thalidomide have been general symptoms such as somnolence and headache, peripheral neuropathy, constipation, skin rash, and other symptoms. Although these adverse effects are generally reversible and mild, grade 3 and 4 toxicities such as peripheral neuropathy, deep venous thrombosis, neutropenia, and toxic dermal necrosis have occasionally been reported. The application of thalidomide therapy in patients with multiple myeloma is being broadened to include not only cases of refractory myeloma, but also previously untreated cases, as well as for maintenance therapy after hematopoietic stem cell transplantation and for the treatment of other hematological diseases. The safe use of this drug will depend on the establishment of diagnostic and treatment guidelines. In addition, the establishment of a nation-wide regulation system is urgently needed in Japan. [source]

Increased Expression of p53 Protein Correlates With the Extent of Myocyte Damage in Cardiac Allograft Rejection

CONGESTIVE HEART FAILURE, Issue 6 2008
Bernadette K. McLaren MD
Acute cardiac allograft rejection (ACAR) has been associated with a poor prognosis. The early diagnosis of ACAR necessitates the accurate detection of myocyte damage. Nuclear damage activates p53, a transcription factor that initiates apoptosis and repair. Endomyocardial biopsies (n=25) from 10 cardiac allograft recipients were stained for nuclear p53. The biopsies were divided into rejection groups based on the grading of ACAR: group 1, grade 0; group 2, grade Ia and Ib; group 3, grades II and III. While clinical indices did not correlate with myocyte damage, significantly more myocytes in group 3 stained for nuclear p53 (2.48±0.60/mm2) compared with group 1 (0.22±0.12/mm2) and group 2 (0.43±0.18/mm2). Increased expression of p53 in cardiac myocytes with grade II or grade III rejection provides an objective quantification as an aid in the diagnosis of ACAR. [source]

Induction of apoptosis by A3 adenosine receptor agonist N6 -(3-iodobenzyl)-adenosine-5,- N -methylcarboxamide in human leukaemia cells: a possible involvement of intracellular mechanism

ACTA PHYSIOLOGICA, Issue 2 2010
P. Mlejnek
Abstract Aim:, The sensitivity of cancer cells which exhibit multi-drug resistance phenotype to A3 adenosine receptor (A3AR) agonist N6 -(3-iodobenzyl)-adenosine-5,- N -methylcarboxamide (IB-MECA) was studied. Methods:, To establish direct relationship between P-glycoprotein (P-gp, ABCB1 and MDR1) expression and IB-MECA induced cell death, a straightforward method for precise estimation of intracellular level of this A3AR agonist was developed. Results:, We subjected three human leukaemia cell lines HL-60, K562 and K562/HHT to treatment with micromolar concentrations of IB-MECA. Although all cell lines used expressed A3AR, there was a large difference in their sensitivity to IB-MECA. While HL-60 and K562 cells were almost equally sensitive, the K562/HHT cells, which exhibit a multi-drug resistance phenotype because of overexpression of P-gp, were significantly more resistant. We found that the intracellular level of IB-MECA in K562/HHT cells was approx. 10 times lower than those in HL-60 or K562 cells. Inhibitors of P-gp, including cyclosporine A (CsA) and verapamil (Vpa), increased the intracellular level of IB-MECA and reversed the resistance of K562/HHT cells to this drug. Accordingly, shRNA-mediated down-regulation of P-gp significantly increased the intracellular level of IB-MECA in K562/HHT cells which simultaneously exhibited reduced resistance to this A3AR agonist. In addition, an in vitro enzyme-based assay provided evidence that IB-MECA might serve as a substrate for P-gp. Conclusion:, Our results suggest that P-gp overexpression prevents cells from IB-MECA induced apoptosis despite the A3AR expression. Pro-apoptotic effect of IB-MECA seemed to strongly depend on its intracellular accumulation rather than on its interaction with A3AR. [source]

Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) and its receptor Fn14 during cardiac remodelling in rats

ACTA PHYSIOLOGICA, Issue 1 2010
E. Mustonen
Abstract Aim:, Accumulating evidence supports the concept that proinflammatory cytokines play an essential role in the failing heart. We examined the concomitant tumour necrosis factor-like weak inducer of apoptosis (TWEAK)/Fn14 expression in myocytes in vitro as well as in vivo in cardiac remodelling. Methods:, We assessed TWEAK and its receptor Fn14 expression in response to angiotensin (Ang) II, myocardial infarction (MI) as well as to local adenovirus-mediated p38 gene transfer in vivo. The effect of various hypertrophic factors and mechanical stretch was studied in neonatal rat ventricular myocyte cell culture. Results:, Ang II increased Fn14 levels from 6 h to 2 weeks, the greatest increase in mRNA levels being observed at 6 h (6.3-fold, P < 0.001) and protein levels at 12 h (4.9-fold, P < 0.01). TWEAK mRNA and protein levels remained almost unchanged during Ang II infusion. Likewise, a rapid and sustained elevation of Fn14 mRNA and protein levels in the left ventricle was observed after experimental MI. Moreover, local p38 gene transfer increased Fn14 mRNA and protein but not TWEAK levels. Fn14 immunoreactive cells were mainly proliferating non-myocytes in the inflammation area while TWEAK immunoreactivity localized to cardiomyocytes and endothelial cells of the coronary arteries. Hypertrophic agonists and lipopolysaccharide increased Fn14 but not TWEAK gene expression in neonatal rat myocytes, while mechanical stretch upregulated Fn14 and downregulated TWEAK gene expression. Conclusions:, In conclusion, the cardiac TWEAK/Fn14 pathway is modified in response to myocardial injury, inflammation and pressure overload. Furthermore, our findings underscore the importance of Fn14 as a mediator of TWEAK/Fn14 signalling in the heart and a potential target for therapeutic interventions. [source]

Orexins/hypocretins and orexin receptors in apoptosis: a mini-review

ACTA PHYSIOLOGICA, Issue 3 2010
M. Laburthe
Abstract An unexpected and fascinating aspect of the neuropeptides orexins has recently emerged when it was shown that orexins acting at orexin receptors OX1R or OX2R induce dramatic apoptosis resulting in massive reduction in cell growth in various cancer cell lines. This mini-review will provide the reader with recent findings related to the proapoptotic actions of orexins and the entirely novel mechanism whereby the seven membrane-spanning G-protein-coupled receptor (GPCR) OX1R triggers apoptosis. Recent data show that orexins induce tyrosine phosphorylation of the tyrosine-based motifs , immunoreceptor tyrosine-based inhibitory motif and immunoreceptor tyrosine-based switch motif , in OX1R. These phosphorylations result in the recruitment and activation of the phosphotyrosine phosphatase SHP-2 and subsequent cytochrome c -mediated mitochondrial apoptosis. Finally, this mini-review will also speculate on: (1) the potential importance of tyrosine-based motifs in the large family of GPCRs; (2) the interest of orexin receptors as therapeutic targets in cancer therapy; (3) the possible role of orexin receptor-mediated apoptosis in physiology and pathophysiology in the brain (neurodevelopment, neurodegenerative diseases) and in the periphery. [source]

Reactive oxygen and nitrogen species in normal physiological processes

ACTA PHYSIOLOGICA, Issue 1 2010
J. Pourova
Abstract Reactive oxygen species (ROS) and reactive nitrogen species have generally been considered as being highly reactive and cytotoxic molecules. Besides their noxious effects, ROS participate in physiological processes in a carefully regulated manner. By way of example, microbicidal ROS are produced in professional phagocytes, ROS function as short-lived messengers having a role in signal transduction and, among other processes, participate in the synthesis of the iodothyronine hormones, reproduction, apoptosis and necrosis. Because of their ability to mediate a crosstalk between key molecules, their role might be dual (at least in some cases). The levels of ROS increase from a certain age, being associated with various diseases typical of senescence. The aim of this review is to summarize the recent findings on the physiological role of ROS. Other issues addressed are an increase in ROS levels during ageing, and the possibility of the physiological nature of this process. [source]

Prolonged exposure to inhaled nitric oxide transiently modifies tubular function in healthy piglets and promotes tubular apoptosis

ACTA PHYSIOLOGICA, Issue 4 2009
W. Go, dzik
Abstract Aim:, Inhaled nitric oxide (iNO) is a selective pulmonary vasodilator. We hypothesized that those piglets exposed to prolonged iNO react with a modified renal function. Methods:, Randomized, placebo-controlled exposure to 40 p.p.m. iNO (30 h) in piglets (n = 20). Plasma and urine were sampled during three periods (first and second 12 h periods, and finally a 6 h period). We measured urine volumes, plasma and urine electrolytes (UNa, UK, UCl), plasma creatinine and urea. We calculated creatinine clearance (Ccr), and fractional excretions of sodium and potassium (FENa, FEK) and urinary excretions of electrolytes (UENa, UEK, UECl). Haemodynamic data were recorded and renal tubular apoptosis detected. Results:, For the first 12 h, certain parameters significantly increased in the iNO group (mean ± SD): UNa (mmol L,1), 87.7 (±35.0) vs. 39.3 (±22.9), UCl (mmol L,1) 80.4 (±32.8) vs. 48.0 (±26.7), FENa (%) 2.1 (±0.8) vs. 0.7 (±0.5), FEK (%) 31.7 (±7.0) vs. 20.7 (±12.3), as well as UENa (mmol) 61.0 (±21.1) vs. 27.6 (±17.9) and UECl (mmol) 57.3 (24.5) vs. 37.6 (29.0). These changes were absent in the second and third periods of the study. Significant differences in percentage of apoptotic cell nuclei in the renal cortex and medulla were found after iNO exposure: 39% vs. 15%. Conclusion:, Exposure to 40 p.p.m. iNO in healthy anaesthetized piglets has a transient natriuretic effect that disappears after 12 h. We also found evidence of renal tubular apoptosis promotion after 30 h of iNO. [source]

When is high-Ca2+ microdomain required for mitochondrial Ca2+ uptake?,

ACTA PHYSIOLOGICA, Issue 1 2009
A. Spät
Abstract Ca2+ release from IP3 -sensitive stores in the endoplasmic reticulum (ER) induced by Ca2+ -mobilizing agonists generates high-Ca2+ microdomains between ER vesicles and neighbouring mitochondria. Here we present a model that describes when such microdomains are required and when submicromolar [Ca2+] is sufficient for mitochondrial Ca2+ uptake. Mitochondrial Ca2+ uptake rate in angiotensin II-stimulated H295R adrenocortical cells correlates with the proximity between ER vesicles and the mitochondrion, reflecting the uptake promoting effect of high-Ca2+ peri-mitochondrial microdomains. Silencing or inhibition of p38 mitogen-activated protein kinase (MAPK) or inhibition of the novel isoforms of protein kinase C enhances mitochondrial Ca2+ uptake and abolishes the positive correlation between Ca2+ uptake and ER-mitochondrion proximity. Inhibition of protein phosphatases attenuates mitochondrial Ca2+ uptake and also abolishes its positive correlation with ER-mitochondrion proximity. We postulate that during IP3 -induced Ca2+ release, Ca2+ uptake is confined to ER-close mitochondria, because of the simultaneous activation of the protein kinases. Attenuation of Ca2+ uptake prevents Ca2+ overload of mitochondria and thus protects the cell against apoptosis. On the other hand, all the mitochondria accumulate Ca2+ at a non-inhibited rate during physiological Ca2+ influx through the plasma membrane. Membrane potential is higher in ER-distant mitochondria, providing a bigger driving force for Ca2+ uptake. Our model explains why comparable mitochondrial Ca2+ signals are formed in response to K+ and angiotensin II (equipotent in respect to global cytosolic Ca2+ signals), although only the latter generates high-Ca2+ microdomains. [source]

The death of cardiotonic steroid-treated cells: evidence of Na+i,K+i -independent H+i -sensitive signalling

ACTA PHYSIOLOGICA, Issue 1-2 2006
S. N. Orlov
Abstract Na/K-ATPase is the only known target of cardiotonic steroids (CTS) identified in plants, amphibians and later on in several mammalian species, including human. We focus our review on recent data implicating CTS in the tissue-specific regulation of cell survival and death. In vascular smooth muscle cells, CTS inhibited cell death triggered by apoptotic stimuli via a novel Na+i -mediated, Ca2+i -independent mechanism of expression of antiapoptotic genes, including mortalin. In contrast, exposure to CTS in vascular endothelial and renal epithelial cells led to cell death, showing combined markers of apoptosis and necrosis. This mode of cell death, termed oncosis, is caused by CTS interaction with Na/K-ATPase but is independent of the inhibition of Na/K-ATPase-mediated ion fluxes and inversion of the [Na+]i/[K+]i ratio. The intermediates of intracellular signalling involved in Na+i, K+i -independent oncosis of CTS-treated cells remain unknown. Recently, we found that this mode of cell death can be protected by modest intracellular acidification via the expression of H+i -sensitive genes. The molecular origin of intracellular Na+ and H+ sensor involvement in the development of apoptosis and oncosis is currently under investigation. [source]

Remission induction chemotherapy induces in vivo caspase-dependent apoptosis in bone marrow acute myeloid leukemia blast cells and spares lymphocytes

CYTOMETRY, Issue 3 2006
J.-P. Vial
Abstract Background The goal of new therapeutic strategies is to adapt the treatment of acute myeloid leukemia (AML) patients to the prognostic and/or to the hematological response. Methods We analyzed in vivo apoptosis induction in blast cells and in lymphocytes of AML patients receiving remission induction treatment. Results We show, on 12 peripheral blood samples, that the increase of peripheral apoptotic blast cells cannot be considered as the earliest marker of the treatment efficiency, because the significant increase of apoptosis followed the white blood cell and the peripheral blast cell count reductions, probably due to an efficient clearance of circulating apoptotic cells. Furthermore, the study of 65 bone marrow samples at d15 showed that the treatment induced apoptosis of blast cells while sparing the lymphocytes. This apoptosis was evidenced both at the caspase and at the membrane levels using respectively fmk-VAD-FITC and Annexin V binding assays. We found that less than 50% of apoptosis, measured with the fmk-VAD-FITC, in the d15 residual bone marrow blast cells, correlated with lower disease-free survival probability. Conclusion More studies are needed in larger series and earlier during the remission induction treatment to confirm the possible prognostic significance of in vivo apoptosis induction. © 2006 International Society for Analytical Cytology [source]

JC-1, a sensitive probe for a simultaneous detection of P-glycoprotein activity and apoptosis in leukemic cells

CYTOMETRY, Issue 3 2006
Driss Chaoui
Abstract Background JC-1 probe has been successfully used for the analysis of either apoptosis or P-glycoprotein (P-gp) activity. Therefore, we wanted to see if JC-1 could also simultaneously assess both, P-gp activity and apoptosis, in acute myeloid leukemia (AML) cells. Methods P-gp activity was measured using JC-1 and compared to the results of the Rhodamine 123 (Rh 123) assay in P-gp negative and P-gp positive cell lines, and 12 AML samples. For apoptosis, spontaneous apoptosis, as well as, apoptosis induced by Cytosine Arabinosine and Homoharringtonine were analyzed. Both mitochondrial red fluorescence and cytoplasmic green fluorescence of JC-1 with and without a P-gp inhibitor (Cyclosporine A : CsA) were used for the identification of apoptotic cells, and this was compared to Annexin V/PI staining. Results (1) We found a good correlation between JC-1 and Rh 123 in viable cells. Even in a small population of viable cells, P-gp positive cells emitting low red fluorescence, gained on red fluorescence after P-gp inhibition with CsA permitting an evaluation of P-gp activity. (2) We found a good correlation between the Annexin V/PI staining and JC-1 (P < 0.0001) in the assessment of apoptotic cells. Most importantly, the apoptotic cells could be distinguished by the loss of red fluorescence and the increase of green fluorescence without any change after P-gp inhibition with CsA. Conclusions JC-1 can simultaneously evaluate two important parameters involved in drug resistance in AML cells, P-gp activity and apoptosis. © 2006 International Society for Analytical Cytology [source]

Spontaneous apoptosis in chronic lymphocytic leukemia and its relationship to clinical and cell kinetic parameters

CYTOMETRY, Issue 6 2001
Gislaine B. Oliveira
Abstract Chronic lymphocytic leukemia (CLL) presents considerable variability in clinical presentation as well as in its evolution. In contrast to the inhibition of apoptosis in vivo, spontaneous apoptosis after short-term culture occurs. We studied the degree of this apoptosis in vitro, and its interactions with several clinical and laboratory parameters. Apoptosis was measured by the annexin V technique. Proliferation rate was evaluated by the AgNOR (nucleolar organizer regions) technique. There were inverse correlations between the percentage of annexin V-positive cells and peripheral lymphocyte count (r = - 0.49), Rai stage (r = - 0.40), Binet stage (r = - 0.50), TTM (total tumor mass score; r = - 0.51), and percentage of cells with one AgNOR cluster (r = - 0.45). Direct correlations were found with hemoglobin values ( r = 0.34) and platelet counts (r = 0.52). The number of CD8-positive cells showed a correlation with peripheral lymphocyte count (r = 0.49). When this variable was held constant, a correlation was detected between CD8-positive cells and staging (r = -0.47), TTM (r = - 0.42), and platelet count (r = 0.67). CD4-positive lymphocytes presented a correlation only with CD8-positive lymphocytes. In a cluster analysis, it was possible to create three groups of patients with different apoptosis rates using the TTM and AgNOR values. We conclude that, with the progression of the disease, together with the increase of tumor mass and proliferation rate, there is a decrease in the suceptibility to apoptosis. Cytometry (Comm. Clin. Cytometry) 46:329,335, 2001. © 2001 Wiley-Liss, Inc. [source]

Treadmill exercise in mice increases intestinal lymphocyte loss via apoptosis

ACTA PHYSIOLOGICA, Issue 3 2003
L. Hoffman-Goetz
Abstract Strenuous exercise is associated with a transient decline in circulating lymphocytes, possibly through increased apoptosis. Intestinal lymphocytes are important effector cells of intestinal immune function but have not been studied in relation to exercise. Aim:, The purpose of the present study was to examine the effect of exercise on intestinal lymphocyte phenotypes and apoptosis. Methods:, Female C57BL/6 mice (n = 112) were randomized to: (1) treadmill running (90 min, 32 m min,1, 8° grade) and killed immediately after exercise, (2) treadmill running and killed 2 h after exercise, (3) treadmill running and killed 24 h after exercise or (4) a non-exercised control condition with exposure to treadmill noise and vibration without running. Results:, Flow cytometry indicated that the total intestinal CD3+T (P < 0.01), CD4+T (P < 0.005), CD8+T (P < 0.05), pan-NK (P < 0.005) and CD19+B (P < 0.05) lymphocytes were significantly lower 24 h after exercise compared with non-exercised controls. Significantly more CD3+T (P < 0.05) and CD8+T (P < 0.05) intestinal lymphocytes stained positive for annexin V, a marker of apoptosis, at 24 h after exercise compared with intestinal lymphocytes from non-exercised controls. Plasma corticosterone and 8-isoprostane concentrations were also significantly higher immediately after exercise compared with other exercise conditions. Conclusion:, Acute strenuous exercise increases intestinal T (CD3+ and CD8+) lymphocyte loss and apoptosis. The extent to which the exercise-induced apoptosis in intestinal lymphocytes is mediated by increased glucocorticoid concentrations in the gastrointestinal tract will require further studies. [source]

A minor ,-tubulin essential for mammalian cell proliferation

CYTOSKELETON, Issue 9 2008
Rajat Bhattacharya
Abstract Mammals use tubulin from multiple genes to construct microtubules. Some genes are expressed in a tissue specific manner, while others are expressed in almost all cell types. ,5-Tubulin is a minor, ubiquitous isoform whose overexpression was recently shown to disrupt microtubules. Using inhibitory RNA, we now report that suppression of ,5 production in both human and hamster cells blocks cell proliferation. Cells depleted of ,5 either trigger the mitotic checkpoint and undergo apoptosis; or they experience a transient mitotic block, a high incidence of lagging chromosomes, and progression into G1 without cytokinesis to become large, flat cells with elevated DNA content. Microtubules appear to be normally organized in cells depleted of ,5, but they are rich in acetylated ,-tubulin indicating that they may be more stable than normal. The results provide the first evidence that a specific isoform of ,-tubulin is required for mitosis. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

Actin stress fiber pre-extension in human aortic endothelial cells

CYTOSKELETON, Issue 4 2008
Lan Lu
Abstract Actin stress fibers (SFs) enable cells to sense and respond to mechanical stimuli and affect adhesion, motility and apoptosis. We and others have demonstrated that cultured human aortic endothelial cells (HAECs) are internally stressed so that SFs are pre-extended beyond their unloaded lengths. The present study explores factors affecting SF pre-extension. In HAECs cultured overnight the baseline pre-extension was 1.10 and independent of the amount of cell shortening. Decreasing contractility with 30 mM BDM or 10 ,M blebbistatin decreased pre-extension to 1.05 whereas increasing contractility with 2 nM calyculin A increased pre-extension to 1.26. Knockdown of ,-actinin-1 with an interfering RNA increased pre-extension to 1.28. None of these affected the wavelength of the buckled SFs. Pre-extension was the same in unperturbed cells as in those in which the actin cytoskeleton was disrupted by both chemical and mechanical means and then allowed to reassemble. Finally, disrupting MTs or IFs did not affect pre-extension but increased the wavelength. Taken together, these results suggest that pre-extension of SFs is determined primarily by intrinsic factors, i.e. the level of actin-myosin interaction. This intrinsic control of pre-extension is sufficiently robust that pre-extension is the same even after the actin cytoskeleton has been disrupted and reorganized. Unlike pre-extension, the morphology of the compressed SFs is partially determined by MTs and IFs which appear to support the SFs along their lengths. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source]

The Drosophila nucleoporin gene nup154 is required for correct microfilament dynamics and cell death during oogenesis

CYTOSKELETON, Issue 8 2007
Maria Giovanna Riparbelli
Abstract The Drosophila nucleoporin gene nup154 is required in both male and female germline for successful gametogenesis. Mutant flies lack differentiated sperm and lay abnormal eggs. We demonstrated that the egg phenotype was associated with specific alterations of the actin cytoskeleton at different stages of oogenesis. Actually, mutant egg chambers displayed an abnormal organization of both subcortical microfilaments and cytoplasmic actin bundles, that led to defective nurse cell dumping. TUNEL analysis also showed that the dumpless phenotype was associated with delayed apoptosis. The nup154 gene product was localized by conventional immunofluorescence microscopy to the nuclear envelope in a distinct punctuate pattern, characteristic of nuclear pore complex components. TEM analysis revealed that the protein was mainly distributed along filamentous structures that extended radially on the nuclear side of the pore, suggesting that Nup154 could be an integral component of the basket filaments associated with the nuclear pore complexes. We propose that Nup154 is necessary for correct nuclear pore complex functions and that the proper regulation of the actin cytoskeleton dynamics strongly relies upon nuclear pore integrity. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Morphological irregularities and features of resistance to apoptosis in the dcp-1/pita double mutated egg chambers during Drosophila oogenesis

CYTOSKELETON, Issue 1 2005
Ioannis P. Nezis
Abstract In the present study, we demonstrate the most novel characteristic morphological features of Drosophila egg chambers lacking both dcp-1 and pita functions in the germline cells. Dcp-1 is an effector caspase and it has been previously shown to play an important role during Drosophila oogenesis [McCall and Steller, 1998 : Science 279 : 230,234; Laundrie et al., 2003 : Genetics 165 : 1881,1888; Peterson et al., 2003 : Dev Biol 260 : 113,123]. The completion of sequencing and annotation of the Drosophila genome has revealed that the dcp-1 gene is nested within an intron of another distinct gene, called pita, a member of the C2H2 zinc finger protein family that regulates transcriptional initiation. The dcp-1,/,/pita,/, nurse cells exhibit euchromatic nuclei (delay of apoptosis) during the late stages of oogenesis, as revealed by conventional light and electron microscopy. The phalloidin-FITC staining discloses significant defects in actin cytoskeleton arrangement. The actin bundles fail to organize properly and the distribution of actin filaments in the ring canals is changed compared to the wild type. The oocyte and the chorion structures have been also modified. The oocyte nucleus is out of position and the chorion appears to contain irregular foldings, while the respiratory filaments obtain an altered morphology. The dcp-1,/,/pita,/, egg chambers do not exhibit the rare events of spontaneously induced apoptosis, observed for the wild type flies, during mid-oogenesis. Interestingly, the mutated egg chambers are protected by staurosporine-induced apoptosis in a percentage of 40%, strongly suggesting the essential role of dcp-1 and/or pita during mid-oogenesis. Cell Motil. Cytoskeleton 60:14,23, 2005. © 2004 Wiley-Liss, Inc. [source]

Remodeling of the actin cytoskeleton of target hepatocytes and NK cells during induction of apoptosis

CYTOSKELETON, Issue 2 2001
W. Marty Blom
Abstract Natural Killer cells are immune cells that recognize and eliminate altered and non-self cells from the circulation. To study the interaction between NK cells and target cells, we set up an experimental system consisting of rat Interleukin-2 activated Natural Killer cells (A-NK cells) and rat hepatocytes with a masked Major Histocompatibility Complex (MHC). The masking of the MHC induces recognition of the hepatocytes by the NK cells as non-self. We showed that in vitro apoptosis is rapidly induced in the hepatocytes [Blom et al., 1999] after co-incubation with A-NK cells. Now we describe the morphological changes that occur during and after interaction of A-NK cells with hepatocytes. Confocal laser scanning microscopy showed that the actin cytoskeleton of the NK cells was remodeled during attack of hepatocytes. Some NK cells were in close contact with the hepatocytes while others had formed actin-containing dendrites of varying length that made contact with the hepatocytes. However, dendrite formation is not obligatory for induction of apoptosis because cells that were unable to form these did induce FAS-dependent apoptosis in hepatocytes. Apparently both direct as well as distant contact resulted in apoptosis. Formation of the dendrites was calcium-dependent as EGTA largely prevented it. Importantly, chelation of the calcium also suppressed killing of the hepatocytes. Within 1 h after addition of the A-NK cells, morphological changes in hepatocytes that are characteristic of apoptosis, such as the formation of apoptotic bodies and fragmented nuclei, became apparent. Specifically, the actin cytoskeleton of the hepatocytes was remodeled resulting in the formation of the apoptotic bodies. Inhibition of caspase activity by z-Val-Ala-DL-Asp-fluoromethylketone (100 ,M) partly protected against the rearrangement of the actin filaments in the hepatocytes. Cell Motil. Cytoskeleton 49:78,92, 2001. © 2001 Wiley-Liss, Inc. [source]

Effect of storage media on human periodontal ligament cell apoptosis

DENTAL TRAUMATOLOGY, Issue 1 2008
Mónica M. Chamorro
However, the mechanisms by which different storage conditions alter the functional status of PDL cells have not been determined. The purpose of the present study was to investigate, in vitro, the level of programed cell death or apoptosis in a population of PDL cells following storage under different conditions. Primary human PDL cells were plated into 24-well-culture plates and allowed to attach for 24 h. Cells were then exposed for 1 h to milk, Hank's balanced salt solution (HBSS), Soft Wear contact lens solution or Gatorade at room temperature or on ice. Culture medium was used as a negative control. Apoptosis was evaluated at 24, 48, and 72 h after treatment on quadruplicate samples by using the ST 160 ApopTag Fluorescein Direct In Situ Detection Kit. The total number of cells and the total number of apoptotic cells were counted. The results indicated that at 24 and 72 h, PDL treated with Gatorade and the contact lens solution displayed the highest percentages of apoptotic cells when compared with the other treatment groups at room temperature. Overall, cells treated on ice showed significantly lower levels of apoptosis when compared with treatments at room temperature. In conclusion, the results indicated that apoptosis plays a major role in cell death in cells treated with Gatorade and contact lens solutions in comparison to other storage solutions and that storage on ice can inhibit programed cell death. [source]

Cryolipolysis for Noninvasive Fat Cell Destruction: Initial Results from a Pig Model

DERMATOLOGIC SURGERY, Issue 10 2009
BRIAN ZELICKSON MD
BACKGROUND Liposuction is one of the most frequently performed cosmetic procedures in the United States, but its cost and downtime has led to the development of noninvasive approaches for adipose tissue reduction. OBJECTIVE To determine whether noninvasive controlled and selective destruction of fat cells (Cryolipolysis) can selectively damage subcutaneous fat without causing damage to the overlying skin or rise in lipid levels. METHODS Three Yucatan pigs underwent Cryolipolysis at 22 sites: 20 at cooling intensity factor (CIF) index 24.5 (,43.8 mW/cm2), one at CIF 24.9 (,44.7 mW/cm2), and one at CIF 25.4 (,45.6 mW/cm2). Treated areas were evaluated using photography, ultrasound, and gross and microscopic pathology. Lipids were at various times points. One additional pig underwent Cryolipolysis at various days before euthanasia. RESULTS The treatments resulted in a significant reduction in the superficial fat layer without damage to the overlying skin. An inflammatory response triggered by cold-induced apoptosis of adipocytes preceded the reduction in the fat layer. Evaluation of lipids over a 3-month period following treatment demonstrated that cholesterol and triglyceride values remained normal. CONCLUSIONS Cryolipolysis is worthy of further study because it has been shown to significantly decrease subcutaneous fat and change body contour without causing damage to the overlying skin and surrounding structures or deleterious changes in blood lipids. [source]

Hydrogen Peroxide and Wound Healing: A Theoretical and Practical Review for Hair Transplant Surgeons

DERMATOLOGIC SURGERY, Issue 6 2008
SARA WASSERBAUER MD
BACKGROUND In most hair restoration practices, hydrogen peroxide has been routinely used to remove blood during and after hair transplant surgery. In other specialties, hydrogen peroxide is also used in these ways: wound cleaning, prevention of infection, hemostasis, and removal of debris. Despite its widespread use, there are still concerns and controversy about the potential toxic effect of hydrogen peroxide. OBJECTIVE The objective was to review all available literature including in vivo and in vitro effects of hydrogen peroxide, as well as general wound healing research. MATERIAL AND METHODS Literature up to and including the past three decades was investigated. RESULTS Two pilot studies were found, and there are not enough data examining the real impact of using hydrogen peroxide in hair transplant surgery. In other specialties, H2O2 appears to have positive effects, such as stimulation of vascular endothelial growth factor, induction of fibroblast proliferation, and collagen, or negative effects, such as cytotoxicity, inhibition of keratinocyte migration, disruption of scarless fetal wound repair, and apoptosis. CONCLUSIONS There are not enough data in hair restoration surgery about the use of hydrogen peroxide, and it is unknown and unclear what the optimum dilution should be. Positive and negative effects were found in other specialties. Further studies are recommended. [source]

Treatment of cutaneous T-cell lymphoma with retinoids

DERMATOLOGIC THERAPY, Issue 5 2006
Chunlei Zhang
ABSTRACT:, Retinoids are biologic regulators of differentiation, proliferation, apoptosis, and immune response. Retinoids (all- trans retinoic acid, 13- cis -retinoic acid, and the synthetic analogs isotretinoin, etretinate, and acitretin) have been used for years as monotherapy and/or in combination for treatment of cutaneous T-cell lymphomas (CTCL). Orally administered bexarotene, the first synthetic highly selective retinoid X receptor retinoid to be approved by the Food and Drug Administration for CTCL, was shown to be active against the cutaneous manifestations of all stages of CTCL. The topical gel formulation was also effective for early cutaneous manifestations of CTCL or as an adjunct to systemic or phototherapy. Use of retinoids in future long-term clinical trials and their eventual application in CTCL regiments will require strategies to decrease the side effects of existing retinoids, identify novel receptor subtype-selective retinoids with better therapeutic index, and explore biologically based synergistic combination therapies with other active agents. [source]

Ectopic germline cells in embryos of Xenopus laevis

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2007
Kohji Ikenishi
Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23,48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44,47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43,48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages. [source]

Busulfan-induced central polydactyly, syndactyly and cleft hand or foot: A common mechanism of disruption leads to divergent phenotypes

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2007
Takuji Naruse
The prevalence of clinical phenotypes that exhibit combinations of central polydactyly, syndactyly, or cleft hand or foot is higher than would be expected for random independent mutations. We have previously demonstrated that maternal ingestion of a chemotherapeutic agent, busulfan, at embryonic day 11 (E11) induces these defects in various combinations in rat embryo limbs. In an effort to determine the mechanism by which busulfan disrupts digital development, we examined cell death by Nile Blue staining and TdT-mediated dUTP nick end labeling (TUNEL) assays; we also carried out whole mount in situ hybridization for fibroblast growth factor-8 (Fgf8), bone morphogenetic protein-4 (Bmp4), and sonic hedgehog (Shh) to examine developmental pathways linked to these defects. In busulfan-treated embryos, diffuse cell death was evident in both ectoderm and mesoderm, peaking at E13. The increased cell death leads to regression of Fgf8 in the apical ectodermal ridge (AER) and Bmp4 and Shh in the underlying mesoderm. The subsequent pattern of interdigital apoptosis and cartilage condensation was variably disrupted. These results suggest that busulfan manifests its teratogenic effects by inducing cell death of both ectoderm and mesoderm, with an associated reduction in tissue and a disruption in the generation of patterning molecules during critical periods of digit specification. [source]

Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx mori

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2006
Vicky E. Mpakou
In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster. [source]

Runx3 controls growth and differentiation of gastric epithelial cells in mammals

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2006
Hiroshi Fukamachi
Runx3 is a transcription factor expressed by gastric epithelial cells. In the Runx3,/, mouse, gastric epithelia exhibited hyperplasia, and epithelial apoptosis was suppressed. By analyzing growth of the epithelial cells in primary culture, we found that Runx3,/, gastric epithelial cells are less sensitive to the growth-inhibitory and apoptosis-inducing activities of TGF-,, suggesting that Runx3 is a major growth regulator of gastric epithelial cells by regulating their response to TGF-,. We also found that Runx3 plays an important role in the control of gastric epithelial differentiation. When subcutaneously implanted into nude mice, Runx3,/, gastric epithelial cells formed tumors in which some cells differentiated into intestinal-type cells. Clonal analysis showed that gastric epithelial cells transdifferentiate into intestinal-type cells in the tumor. Considering that gastric epithelial differentiation is very stable, and that intestinal-type cells never differentiate in the mouse stomach, it is remarkable that gastric epithelial cells transdifferentiate into intestinal-type cells. We conclude that Runx3 is deeply involved in the control of both growth and differentiation of gastric epithelial cells. The role of Runx3 in the specification of gastric epithelial cells is discussed. [source]

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2005
Linping Wang
Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15INK4b), and Gas3/PMP22. The expression of p21 and p15INK4b contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death. [source]

Developmental delay and unstable state of the testes in the rdw rat with congenital hypothyroidism

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2004
Yasuhiro Sakai
From the present study of the rdw rat, it is clear that the thyroid hormone is essential for the development and maintenance of the testes. In previous studies, the thyroid hormone has few serious effects on the testes except during the neonatal stage when the thyroid hormone receptor is mainly present. However, there is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span. In the present study, a morphological analysis was performed on the testes of rdw rats with congenital hypothyroidism. The rdw testes required a longer time to develop into the normal adult structure. Moreover, the developed, normal structure began to degenerate after full maturation. Specific characteristics of the rdw testes include: (i) a prolonged proliferation of Sertoli cells during postnatal development; (ii) a developmental delay in the appearance of spermatocytes and spermatid; (iii) direct contact with each other for both spermatocytes and spermatids, without Sertoli cell cytoplasm completely intervening between adjacent germ cells; (iv) subsequent apoptosis of germ cells after maturation; (v) reduction in the height of the seminiferous epithelium; and (vi) lower testosterone levels in the rdw rats, especially during old age. Thus, we conclude that the thyroid hormone plays an important role in developing and maintaining normal function of testes. [source]