Abstract: Autophagy is a physiological and evolutionarily conserved process maintaining homeostatic functions, such as protein degradation and organelle turnover. Accumulating data provide evidence that autophagy also contributes to cell death under certain circumstances, but how this is achieved is not well known. Herein, we report that autophagy occurs during developmentally-induced cell death in the female germline, observed in the germarium and during middle developmental stages of oogenesis in Drosophila melanogaster. Degenerating germline cells exhibit caspase activation, chromatin condensation, DNA fragmentation and punctate staining of mCherry-DrAtg8a, a novel marker for monitoring autophagy in Drosophila. Genetic inhibition of autophagy, by removing atg1 or atg7 function, results in significant reduction of DNA fragmentation, suggesting that autophagy acts genetically upstream of DNA fragmentation in this tissue. This study provides new insights into the mechanisms that regulate cell death in vivo during development.
Abstract: Doxorubicin is an important component of combination therapy for muscle-invasive urinary bladder cancer. Treatment with this topoisomerase II poison is able to interfere with cell cycle progression and lead to cancer cell death. Using FACS analysis, Western immunoblotting and semi-quantitative RT-PCR, we studied the effects of doxorubicin on cell cycle progression and apoptosis, and also explored the possibility of using groups of genes as biomarkers of prognosis and/or response to doxorubicin treatment in human urinary bladder cancer cells. Doxorubicin induced dose-dependent G2/M and/or G1/S cell cycle arrest, followed by grade- and dose-dependent reduction in the amount of the cytosolic trimeric form of FasL, activation of Caspase-8, Caspase-9, Caspase-3, cleavage of PARP, Lamin A/C, Bcl-XL/S and interestingly Hsp90, and finally cell death. Data presented here also suggest the use of the expression patterns of Cyclin-E2, Cyclin-F, p63, p73, FasL, TRAIL, Tweak, Tweak-R, XAF-1, OPG and Bok genes for identification of the differentiation grade, and Cyclin-B2, GADD45A, p73, FasL, Bik, Bim, TRAIL, Fas, Tweak-R, XAF-1, Bcl-2, Survivin, OPG, DcR2 and Bcl-XL genes for the detection of response to doxorubicin in human bladder cancer cells.
Abstract: PURPOSE: Secretory clusterin (sCLU)/apolipoprotein J is an extracellular chaperone that has been functionally implicated in DNA repair, cell cycle regulation, apoptotic cell death, and tumorigenesis. It exerts a prosurvival function against most therapeutic treatments for cancer and is currently an antisense target in clinical trials for tumor therapy. However, the molecular mechanisms underlying its function remained largely unknown. EXPERIMENTAL DESIGN: The molecular effects of small interfering RNA-mediated sCLU depletion in nonstressed human cancer cells were examined by focusing entirely on the endogenously expressed sCLU protein molecules and combining molecular, biochemical, and microscopic approaches. RESULTS: We report here that sCLU depletion in nonstressed human cancer cells signals stress that induces p53-dependent growth retardation and high rates of endogenous apoptosis. We discovered that increased apoptosis in sCLU-depleted cells correlates to altered ratios of proapoptotic to antiapoptotic Bcl-2 protein family members, is amplified by p53, and is executed by mitochondrial dysfunction. sCLU depletion-related stress signals originate from several sites, because sCLU is an integral component of not only the secretory pathway but also the nucleocytosolic continuum and mitochondria. In the cytoplasm, sCLU depletion disrupts the Ku70-Bax complex and triggers Bax activation and relocation to mitochondria. We show that sCLU binds and thereby stabilizes the Ku70-Bax protein complex serving as a cytosol retention factor for Bax. CONCLUSIONS: We suggest that elevated sCLU levels may enhance tumorigenesis by interfering with Bax proapoptotic activities and contribute to one of the major characteristics of cancer cells, that is, resistance to apoptosis.
Abstract: It is increasingly recognized that programmed cell death includes not only apoptosis and autophagy, but also other types of nonapoptotic cell death, such as paraptosis, which are all characterized by distinct morphological features. Our findings indicate that all three types of programmed cell death occur in the ovarian nurse cell cluster during late vitellogenesis (formation of the egg yolk) of Bombyx mori (Lepidoptera), whereas middle vitellogenesis is exclusively characterized by the presence of a nonapoptotic type of cell death, known as paraptosis. During middle vitellogenesis, nurse cells exhibit clearly cytoplasmic vacuolization, as revealed by ultrastructural examination performed through conventional light and transmission electron microscopy, while no signs of apoptotic or autophagic features are detectable. Moreover, nurse cells of developmental stages 7, 8 and 9 contain autophagic compartments, as well as apoptotic characteristics, such as condensed chromatin, fragmented DNA and activated caspases, as revealed by in vitro assays. We propose that paraptosis precedes both apoptosis and autophagy during vitellogenesis, since its initial activation is detectable during middle vitellogenesis, whereas no apoptotic nor autophagic features are observed. In contrast, at the late stages of Bombyx mori oogenesis, paraptosis, autophagy and apoptosis operate synergistically, resulting in a more efficient elimination of the degenerated nurse cells.
Abstract: Oogenesis is a fundamental physiological process in insects. Successful oogenesis is critical for evolutionary success by transferring genetic information to the next generation. This is achieved by the normal maturation of the egg chamber (egg), which is accomplished through cell death of the cells that accompany the oocyte. Recent studies demonstrate that autophagy contributes to this cell death process. Hence, comprehension of the mechanisms that implicates autophagy during cell death in insect eggs is very important. Herein, we describe some experimental approaches that can be used to monitor autophagy in insect eggs.
Abstract: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
Abstract: Familial Hypertrophic Cardiomyopathy (FHC) constitutes a genetic disease of the sarcomere characterized by a Mendelian pattern of inheritance. A variety of different mutations affecting the at least eight sarcomeric gene products has been identified and the majority of them appear to function through a dominant negative mechanism. Family history analysis and genetic counseling have been widely adopted as integral tools for the evaluation and management of individuals with Hypertrophic Cardiomyopathy (HCM). Genetic testing of the disease has been progressively released into the clinical mainstream, thus rendering the development of novel and potent molecular diagnostic protocols an inevitable task. To this direction, we have evolved an integrated PCR-based molecular protocol, which through the utilization of novel "exonic" primers allows, among others, the structural analysis of the 13th exon of the human beta-myosin heavy chain gene locus (MYH7) mainly characterized by the critical for HCM Arginine residue 403 (R(403)). Interestingly, through a DNA sequencing approach, a single nucleotide substitution from "G" to "T" was detected in the adjacent 13th intron, thus divulging the versatile potential of the present molecular protocol to clinical practice.
Abstract: BACKGROUND: Red cells (RBCs) lose membrane in vivo, under certain conditions in vitro, and during the ex vivo storage of whole blood, by releasing vesicles. The vesiculation of the RBCs is a part of the storage lesion. The protein composition of the vesicles generated during storage of banked RBCs has not been studied in detail. STUDY DESIGN AND METHODS: Vesicles were isolated from the plasma of nonleukoreduced RBC units in citrate-phosphate-dextrose-adenine, at eight time points of the storage period and shortly afterward. The degree of vesiculation, ultrastructure, oxidation status, and protein composition of the vesicles were evaluated by means of electron microscopy and immunoblotting. RBCs and ghost membranes were investigated as controls. RESULTS: The total protein content of the vesicle fraction and the size of the vesicles increased but their structural integrity decreased over time. The oxidation index of the vesicles released up to Day 21 of storage was greater than that of the membrane ghosts of the corresponding intact RBCs. The vesicles contain aggregated hemoglobin, band 3, and lipid raft proteins, including flotillins. They also contain Fas, FADD, procaspases 3 and 8, caspase 8 and caspase 3 cleavage products (after the 10th day), CD47 (after the 17th day), and immunoglobulin G. CONCLUSION: These data indicate that the vesicles released during storage of RBCs contain lipid raft proteins and oxidized or reactive signaling components commonly associated with the senescent RBCs. Vesiculation during storage of RBCs may enable the RBC to shed altered or harmful material.
Abstract: Programmed cell death consists of two major types, apoptotic and autophagic, both of which are mainly defined by morphological criteria. Our findings indicate that both types of programmed cell death occur in the ovarian nurse cells during middle- and late-oogenesis of Drosophila virilis. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain condensed chromatin and fragmented DNA, whereas active caspase assays and immunostaining procedures demonstrate the presence of highly activated caspases. Distinct features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an accumulation of lysosomal proteases. At the late stages of D. virilis oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones described above, being also escorted by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during D. virilis oogenesis for a more efficient elimination of the degenerated nurse cells.
Abstract: We describe the features of programmed cell death occurring in the egg chambers of Drosophila virilis during mid-oogenesis and late oogenesis. During mid-oogenesis, the spontaneously degenerating egg chambers exhibit typical characteristics of apoptotic cell death. As revealed by propidium iodide, rhodamine-conjugated phalloidin staining, and the TUNEL assay, respectively, the nurse cells contain condensed chromatin, altered actin cytoskeleton, and fragmented DNA. In vitro caspase activity assays and immunostaining procedures demonstrate that the atretic egg chambers possess high levels of caspase activity. Features of autophagic cell death are also observed during D. virilis mid-oogenesis, as shown by monodansylcadaverine staining, together with an ultrastructural examination by transmission electron microscopy. During the late stages of oogenesis in D. virilis, once again, the two mechanisms, viz., nurse cell cluster apoptosis and autophagy, operate together, manifesting features of cell death similar to those detailed above. Moreover, an altered form of cytochrome c seems to be released from the mitochondria in the nurse cells proximal to the oocyte. We propose that apoptosis and autophagy function synergistically during oogenesis in D. virilis in order to achieve a more efficient elimination of the degenerated nurse cells and abnormal egg chambers.
Abstract: AIMS: To investigate the clinicopathological and prognostic significance of membrane type 1 matrix metalloproteinase (MT1-MMP) and MMP-9 proteins expression in invasive breast carcinoma and their relationship to tumour proliferation and expression of c-erbB2 and peroxisome proliferator-activated receptor (PPAR) gamma. METHODS: Immunohistochemistry was carried out on 175 paraffin-embedded breast tissue specimens to detect MT1-MMP, MMP-9, oestrogen receptor (ER), progesterone receptor, c-erbB-2, Ki67, topoisomerase IIalpha (topo IIalpha) and PPARgamma protein expression. RESULTS: Both MT1-MMP and MMP-9 were expressed in the cytoplasm of the malignant cells and the peritumoral stroma. Cytoplasmic MT1-MMP was more often observed in ER+ tumours (P = 0.022), of a lower nuclear grade (P = 0.020) and with reduced expression of Ki67 and topo IIalpha (P = 0.027 and P = 0.006, respectively). Moreover, cytoplasmic MT1-MMP was positively associated with MMP-9 (P = 0.010) and PPARgamma (P < 0.0001). Cytoplasmic MMP-9 was inversely associated with Ki67 (P = 0.034) and topo IIalpha (P = 0.004), whereas its relationship with MT1-MMP (P = 0.034) and PPARgamma (P = 0.024) was found to be positive. Stromal MMP-9 was more often observed in c-erbB2+ tumours (P = 0.043) and had an unfavourable impact on overall and relapse-free survival in both univariate (P = 0.0157 and P = 0.0274, respectively) and multivariate analyses (P = 0.007 and P = 0.024, respectively). CONCLUSIONS: Cytoplasmic MT1-MMP and MMP-9 seem to be related to well-differentiated tumours, with a low proliferation potential, while stromal MMP-9 is associated with an aggressive tumour phenotype and is recognized as an independent poor prognostic indicator.
Abstract: In the present study, we describe novel features of programmed cell death in developing egg chambers occurring during mid- and late-oogenesis of the medfly Ceratitis capitata. During mid-oogenesis, the spontaneously degenerated egg chambers exhibit typical characteristics of apoptotic cell death. Their nurse cells contain fragmented DNA and fragmented actin, as revealed by TUNEL assay and immunolabelling, respectively. In vitro caspase activity assays and immunostaining procedures demonstrated that the atretic egg chambers acquired high levels of caspase activity. Distinct features of autophagic cell death were also observed during C. capitata mid-oogenesis, as revealed by the monodansylcadaverine staining approach and ultrastructural examination performed by transmission electron microscopy. Additionally, atretic egg chambers exhibit an upregulation of lysosomal proteases, as demonstrated by a procathepsin L immunolabelling procedure. At the late stages of C. capitata oogenesis, apoptosis and autophagy coexist, manifesting cell death features that are similar to the ones mentioned above, being also chaperoned by the involvement of an altered cytochrome c conformational display. We propose that apoptosis and autophagy operate synergistically during C. capitata oogenesis for a more efficient elimination of the degenerated nurse cells and abnormal egg chambers.
Abstract: Red blood cell (RBC) membrane proteins undergo progressive pathological alterations during storage. In conditions of increased cellular stress, the cytoskeleton also sustains certain modifications. The hemoglobin (Hb) content and oxidative status of the RBC cytoskeletons as a function of the storage period remain unclear. The possible Hb content and oxidative alterations occurring in the cytoskeletons in the course of storage were monitored in six units, by means of electrophoresis, immunoblotting and protein carbonylation assays. A proportion of the ghost-bound Hb consists of non-reducible crosslinkings of probably oxidized(denatured Hb or hemichromes.The defective Hb-membrane association was strongly affected by the prolonged storage. A progressive accumulation of Hb monomers, multimers and high molecular weight aggregates to corresponding cytoskeletons were also evident. The oxidative index of the cytoskeletal proteins was found increased, signalizing oxidative modifications in spectrin and possibly other cytoskeletal proteins. The reported data corroborate the evidence for oxidative damage in membrane proteins with emphasis to the cytoskeletal components. They partially address the pathophysiological mechanisms underlying the RBC storage lesion, add some new insight in the field of RBC storage as a hemoglobin- and cytoskeleton-associated pathology and suggest the possible use of antioxidants in the units intended for transfusion.
Abstract: BACKGROUND: The elucidation of the storage lesion is important for the improvement of red blood cell (RBC) storage. Ex vivo storage is also a model system for studying cell-signaling events in the senescence and programmed cell death of RBCs. The membrane hosts critical steps in these mechanisms and undergoes widespread remodeling over the storage period. STUDY DESIGN AND METHODS: Fresh and CPDA-stored RBCs from 21 blood donors were evaluated as whole cells, membrane ghosts, and cytoskeletons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, immunofluorescence microscopy, and in situ assays. Band 3 content, immunoglobulin G (IgG) content, specific protein movement to and from the membrane, and caspase system activation were measured. RESULTS: During storage, Band 3 protein was aggregated and its content decreased as did the content of several lipid raft-related proteins. IgG binding to the membrane increased. Sorcin and synexin moved from the cytosol to the membrane, stomatin and flotillins left the membrane, the Fas protein was oligomerized, and caspase was activated. CONCLUSION: The remodeling of the RBC membrane during storage includes loss and oxidative cross-linking of Band 3 as well as IgG binding. This process occurs with lipid raft development and loss and is probably driven by caspase activation. Oxidative injury appears to be an important driver of RBC aging during storage.
Abstract: Hereditary spherocytosis (HS) is a heterogeneous group of disorders. The abnormal red cell morphology (resulting in shortened cell survival) is due to a primary deficiency in spectrin, ankyrin-1, band 3 or protein 4.2. Secondary protein deficiencies are often observed and may be involved in the outcome of the disease. In the present study, we searched for secondary erythrocyte membrane protein alterations in HS, including the lipid raft associated proteins and the oxidative index. For this purpose, 12 patients with clinical and laboratory diagnosis of mild to typical HS were examined. Erythrocyte membrane ghosts and skeletons were subjected to SDS-PAGE and immunoblotting analysis using antibodies against red cell membrane proteins and DNP moiety, after 2,4-dinitrophenylhydrazine derivatization. Protein deficiencies, degradation, aggregation and enhanced binding of cytoplasmic components, band 8, hemoglobin and immunoglobulins G to the membrane as well as increased oxidative index, were found in the majority of the HS patients. Proportion of the membrane- and skeleton-bound globin was oxidized/denatured Hb or hemichromes and crosslinkings. Some HS membranes are deficient in lipid rafts proteins and contain sorcin. A context of these distortions is more pronounced in typical HS cases compared to the mild ones. Similar defects in thalassemia and senescent RBCs are dictated by increased oxidative stress and are positively correlated with perturbations in membrane properties. These data add some new insight in the field of HS pathophysiology and clinical variability.
Abstract: The major sites of energy storage during oogenesis in the Drosophila melanogaster oocyte are the alpha- and beta-yolk spheres. By applying biochemical and transmission electron microscopy (TEM) immunogold techniques we found that the beta-yolk spheres contain mainly polysaccharides, while the three main yolk proteins (YPs) are stored in the alpha-yolk spheres of the developing oocyte. Moreover, by using high-resolution TEM of freeze fractured or cryosectioned follicles, we identified the existence of crystalline structures within the alpha-yolk spheres of the mature oocyte. Our subsequent two-dimensional reconstruction analysis revealed that the unit cell of the crystal is about 113 Angstrom x 113 Angstrom. Assuming that the repeating unit is a cylinder of about 110 Angstrom in length and 25 Angstrom in diameter this cylinder would then have a volume of about 50,000 cubic Angstrom, which corresponds to about 40 kDa of protein. This size fits quite well with the known molecular weight of about 40-45 kDa for each of the three D. melanogaster YPs. Overall, our study identifies for the first time the supramolecular arrangement of the alpha-yolk spheres constituent molecules and provides direct evidence for the "natural" crystallization, and therefore the efficient packaging, of the YPs during oogenesis.
Abstract: BACKGROUND: Several rheological disorders of the erythrocytes, such as increased aggregation and decreased deformability, have been observed in diabetes mellitus and have been implicated in the development of diabetic microangiopathy. Structural alterations of the erythrocyte membrane proteins caused by the diabetic process may be at the origin of those observations. In the present study, we searched for erythrocyte membrane protein alterations in diabetic retinopathy. METHODS: We examined peripheral blood samples from 40 type-2 diabetic patients with diabetic retinopathy of variable severity (19 males and 21 females, mean age 66.8 years, Group A) and we compared them with samples from 19 type-2 diabetic patients without diabetic retinopathy (13 males and six females, mean age 66.5 years, Group B) and 16 healthy volunteers (eight males and eight females, mean age 65.6 years, Group C). Erythrocyte membrane ghosts from all samples were subjected to SDS-PAGE, and the electrophoretic pattern of transmembrane and cytoskeletal proteins was analysed for each sample. The protein quantification of each electrophoretic band was accomplished through scanning densitometry. RESULTS: No significant deviations from normal electrophoresis were observed in Groups B and C, apart from an increase in band 8 in two samples from Group B (11%). In contrast, in 14 samples from Group A (35%) we detected increases in protein band 8 and/or membrane-bound haemoglobin along with a decrease in spectrin. Moreover, increased mobility of band 3, an aberrant high molecular weight (MW) (> 255 kDa) band and a low MW (42 kDa) band were evident in ten samples from Group A (25%). Glycophorins were altered in 46% of Group-A patients versus 38% of Group-B patients. Females and patients with long duration of diabetes presented more electrophoretic abnormalities. CONCLUSIONS: Structural alterations of the erythrocyte membrane proteins are shown for the first time in association with diabetic retinopathy. Their detection may serve as a blood marker for the development of diabetic microangiopathy. Further studies are needed to assess whether pharmaceutical intervention to the rheology of erythrocytes can prevent or alleviate microvascular diabetic complications.
Abstract: Programmed cell death, constitutes a common fundamental incident that occurs during oogenesis in a variety of different animals. It plays a significant role in the maturation process of the female gamete and also in the removal of abnormal and superfluous cells at certain checkpoints of development. In the present study, we demonstrate the existence of follicular atresia during mid-oogenesis in the olive fruit fly Dacus oleae (Tephritidae). The number of atretic follicles increases following the age of the fly, suggesting for the presence of an age-susceptible process. The atretic follicles contain nurse cells that exhibit chromatin condensation, DNA fragmentation and actin cytoskeleton alterations, as revealed by propidium iodide staining, TUNEL labeling and phalloidin-FITC staining. Conventional light and electron microscopy disclose that the nurse cell remnants are phagocytosed by the adjacent follicle cells. The follicular epithelium also eliminates the oocyte through phagocytosis, resulting to an egg chamber with no compartmentalized organization. The data presented herein are very similar compared to previous reported results in other Diptera species, strongly suggesting the occurrence of a phylogenetically conserved mechanism of follicular atresia. All these observations also support the notion that mid-oogenesis in D. oleae may be the critical regulation point at which superfluous and defective egg chambers are selectively eliminated before they reach maturity.
Abstract: In the present study, we describe the features of programmed cell death of ovarian follicle cells, occurring during the late developmental stages of oogenesis in the olive fruit fly, Bactrocera oleae and the medfly, Ceratitis capitata. During stage 14, the follicle cells contain autophagic vacuoles, and they do not exhibit caspase activity in all parts of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high- but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. These data argue for the presence of an autophagy-mediated cell death program in the ovarian follicle cell layer in both species. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. We strongly believe that during the termination of the above Dipteran oogenesis, an efficient mechanism of absorption of the degenerated follicle cells is selectively activated, in order to prevent the blockage of the ovarioles and thus robustly support the physiological completion of the ovulation process.
Abstract: Transfusion of allogeneic blood products is associated with adverse reactions and complications. Some of the negative effects of RBC transfusion are associated with the storage lesion. The importance of RBC oxidative damage in the storage lesion is not well documented. We monitored the storage-induced membrane protein oxidation in CPDA-preserved non-leukodepleted RBCs units from five blood donors in the course of the storage period, as assessed by protein carbonylation levels estimation. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and SDS-polyacrylamide gel electrophoresis coupled with Western blotting. Immunoblotting with dinitrophenol-specific antibody revealed increased RBC membrane protein carbonyls with prolonged storage in CPDA units. This finding supports the idea of oxidation as a part of the storage lesion.
Abstract: In the present study, we reveal for the first time that the Bactrocera oleae chorion peroxidase (bPxd) participates essentially in B. oleae chorion formation and clearly represents the homologous member of Drosophila melanogaster chorion peroxidase (Pxd). Comparative sequence analysis disclosed that the bPxd cDNA semi-central region, which encodes for the putative catalytic domain of the enzyme, exhibits great homology (98%) with its Pxd counterpart. Thus, it is very likely that bPxd is highly responsible for the chorion hardening process, through protein cross-linking mediated by the formation of di- and tri-tyrosine bonds. Distinct molecular weight bPxd RNA transcripts were detected in Northern blotting analysis of total RNA extracts of adult flies (2.9 and 1.7 kb) and ovaries (2.2 kb). The ovarian-specific bPxd RNA transcript is selectively expressed in the follicle cell layer during the late stages of oogenesis 12-14, as revealed by in situ hybridization. Moreover, reverse transcription reactions confirmed the stage-specific developmental regulation of the bPxd gene, which is maximally expressed during stage 13. Western blotting with the rabbit anti-rAePO polyclonal antibody revealed three immunoreactive bands of 76, 66 and 54 kDa in crude protein extracts from adult flies, while in larva and purified chorion preparations, a unique 54 kDa band was clearly detected. Immunolocalization experiments revealed that bPxd peroxidase constitutes an essential structural chorionic component, being abundantly localized in all the successive chorionic layers and vitelline membrane as well.
Abstract: Autophagy is a major pathway for the degradation of long-lived proteins and cytoplasmic organelles and an essential part of programmed cell death, as well. Our findings indicate that programmed cell death of the ovarian follicle cells in the higher Diptera species Bactrocera oleae and Ceratitis capitata manifests features of autophagic cell death. The follicle cells during the developmental stage 14 contain autophagic vacuoles and they do not exhibit caspase activity in any area of the egg chamber. Their nuclei are characterized by condensed chromatin, accompanied with high-but not low-molecular weight DNA fragmentation events exclusively detected in distinct cells of the anterior pole. The above results are likely associated with the abundant phagocytosis observed at the entry of the lateral oviducts, where numerous cell bodies are massively engulfed by epithelial cells. The similarity of the cell death process among B. oleae, C. capitata and Drosophila melanogaster species strongly suggests that autophagy-mediated cell death is conserved in higher Diptera species.
Abstract: 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.
Abstract: Programmed cell death constitutes a common fundamental incident occurring during oogenesis in a variety of different organisms. In Drosophila melanogaster, it plays a significant role in the maturation process of the egg chamber. In the present study, we have used an in vitro development system for studying the effects of inducers and inhibitors of programmed cell death during the late stages of oogenesis. Treatment of the developing egg chambers with two widely used inducers of cell death, etoposide and staurosporine, blocks further development and induces chromatin condensation but not DNA fragmentation in nurse and follicle cells, as revealed by propidium iodide staining and terminal transferase-mediated dUTP nick-end labeling assay. Moreover, incubation of the developing egg chambers with the caspase-3 inhibitor Z-DEVD-FMK significantly delays development, prevents DNA fragmentation, but does not affect chromatin condensation. The above results demonstrate, for the first time, that chromatin condensation in Drosophila ovarian nurse and follicle cells is a caspase-3-like independent process and is regulated independently from DNA fragmentation.
Abstract: A full-length cDNA clone, designated Udp1, was isolated from Urtica dioica (stinging nettle), using a polymerase chain reaction based strategy. The putative Udp1 protein is characterized by a cleavable N-terminal signal sequence, likely responsible for the rough endoplasmic reticulum entry and a 310 amino acids mature protein, containing all the important residues, which are evolutionary conserved among different members of the plant peroxidase family. A unique structural feature of the Udp1 peroxidase is defined into the short carboxyl-terminal extension, which could be associated with the vacuolar targeting process. Udp1 peroxidase is differentially regulated at the transcriptional level and is specifically expressed in the roots. Interestingly, wounding and ultraviolet radiation stress cause an ectopic induction of the Udp1 gene expression in the aerial parts of the plant. A genomic DNA fragment encoding the Udp1 peroxidase was also cloned and fully sequenced, revealing a structural organization of three exons and two introns. The phylogenetic relationships of the Udp1 protein to the Arabidopsis thaliana peroxidase family members were also examined and, in combination with the homology modelling approach, dictated the presence of distinct structural elements, which could be specifically involved in the determination of substrate recognition and subcellular localization of the Udp1 peroxidase.
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.
Abstract: In the present study, we demonstrate the isolation and characterization of the Pxd cDNA clone, which codes for the Drosophila melanogaster chorion peroxidase. This specific peroxidase is involved in the chorion hardening process, through protein crosslinking mediated by the formation of di- and tri-tyrosine bonds. The Pxd gene product has been identified in crude protein extracts from adult flies as three immunoreacting, with the anti-rAePO polyclonal antibody, bands of 77, 67 and 55 kDa, while in larvae and purified chorions as a unique 55 kDa band. Moreover, the mature form of the Pxd recombinant protein was specifically recognized by the anti-rAePO antibody as a 77 kDa band, while in the presence of H2O2 was able to convert tyrosine residues to di-tyrosine moieties. Northern blotting analysis of total RNA preparations revealed distinct molecular weight patterns of the Pxd RNA transcripts among adult flies, ovaries and larvae. The in situ hybridization clearly shows that the Pxd mRNA is specifically expressed in follicle cells during the late stages of oogenesis 11-14, while the reverse transcription reactions dictate the stage-specific developmental regulation of the Pxd gene. The immunolocalization approach, using the anti-rAePO polyclonal antibody, has revealed that the Pxd peroxidase is selectively localized in the chorion structures and particularly in the endochorion and innermost chorionic layer (ICL).
Abstract: Congenital dyserythropoietic anemia type I (CDA-I) is a rare genetic disease that affects erythropoiesis. On the other hand, hemoglobin H (HbH) disease is a severe form of alpha-thalassemia. We herein present ultrastructural and immunocytochemical data concerning the first reported case of congenital anemia with clinical and molecular diagnosis of HbH disease complicated by CDA-I-specific dysplasies of the erythroid cells. Fine structure and transmission electron microscope immunolabeling analysis of the bone marrow and peripheral blood samples were consistent with a potential co-existence of the two defects in the same patient, producing a novel and diagnostically important dyserythropoietic profile. In the patient under investigation both nuclear and plasma membrane of the erythroid cells are almost equally defective. The unknown defect causes the concomitant precipitation of beta- and alpha-globin chains (or hemoglobin), along with an unidentified protein(s). The unusual inclusions gain access to the euchromatin area and exhibit higher affinity for the plasma membrane than the classic inclusions of precipitated alpha- or beta-globin chains seen in thalassemia. The affected erythroid precursors are presented with severe nuclear distortions, endonuclear globin loads, morphological evidence of apoptosis and increased erythrophagocytosis. Plasma membrane distortions and the rate of protein precipitation were aggravated with differentiation. Our findings provide additional evidence for a specific activation of a beta-thalassemic-like mechanism in CDA-I, containing not only the hemoglobin biosynthesis as previously suggested, and interpret the prototypal hematological portrait, which is an HbH disease, modified and partially counterbalanced by the effect of CDA-I or an unidentified CDA-I-like disease. The reported data describe the complexity of the interactions between the CDA-I and the HbH disease, revealing essential pathogenic events of the novel anemia and, indirectly, of the CDA-I.
Abstract: In the present paper, we demonstrate the erythroid cell membrane unique properties in a previously characterized case of hemoglobin-H disease, associated with congenital dyserythropoietic anemia type-I features. In order to explain the patient's cell membrane distortions and the high affinity for the various intracellular inclusions, we studied its composition and structure in comparison to other anemic and non-anemic cases. Red cells from peripheral blood were fractionated into cellular, membrane and protein extracts. Membrane attached immunocomplexes were separated and collected by immunoprecipitation. The subcellular fractions were analyzed by SDS-PAGE electrophoresis and immunoblotted against a variety of erythroid-specific antibodies. The protein composition of the membrane was characterized by immunogold electron microscopy. In the membrane of the CDA-associated case, we identified sialic acid and protein deficiencies, formation of protein crosslinkings, excesses of bound globin and immunoglobulins and aberrant peptides. In contrast to the typical hemoglobin-H disease, the ghost-bound globin exhibited preferential attachment to the skeletal proteins than the band 3 and the skeleton-bound globin consisted not only of beta- but also of alpha-globin chains. Another hallmark, probably associated with the CDA defect, was the participation of glycophorins in the membrane-bound immunocomplexes and the pathological clustering of the latter in the membrane. This study strongly suggests that the result of the combinatorial effects on the diseased membrane created a unique profile, quite distinct from the one observed in several typical hemoglobinopathies. Our observations shed light into critical membrane alterations leading to hemolysis in the novel CDA-associated disease and probably into the CDA-I or CDA-I-like diseases.
Abstract: The developmental aspects of the Leptinotarsa decemlineata crystalline chorionic layer (CCL) morphogenesis, its composition and its supramolecular structure were studied. The mature Leptinotarsa decemlineata eggshell consists of the vitelline membrane and the CCL, while the follicle cell remnants following their degeneration after oogenesis completion constitute the outer chorionic layer. The vitelline membrane and the CCL layers are formed through continuous material deposition from the follicular epithelium, whereas the main morphogenic factor during most insect eggshell formation, namely the follicle cell and oocyte microvilli, are seemingly involved only in vitelline membrane formation. Analysis of the CCL morphogenesis showed that this layer is assembled from a fiber-like pre-crystalline material, which accumulates at the vitelline membrane-follicle cell interface. The mature CCL is about 1 microm thick and exhibits a periodicity of approximately 10 nm, while computer image analysis studies of thin-sectioned CCL revealed the existence of crystalline layers parallel to the CCL surface. Finally, SDS-PAGE-electrophoresis of purified CCLs showed that this crystalline layer is of a proteinaceous nature and is most likely composed of 3-5 polypeptides with a molecular weight ranging in between 28-60 kDa. Overall, these data exemplify for the first time the nature and supramolecular arrangement of a crystalline layer and its constituent molecules in Coleoptera.
Abstract: In the present study, we demonstrate the existence of two distinct apoptotic patterns in nurse cells during Ceratitis capitata oogenesis. One is developmentally regulated and normally occurs during stages 12 and 13, and the other is stage specific and is sporadically observed during stages 7 and 8. The pre-apoptotic manifestation of the first pattern begins at stage 11 and is characterized by the formation of actin bundles. Subsequently, at stages 12 and 13, the nurse cell nuclei exhibit condensed chromatin and contain fragmented DNA, as revealed by TUNEL assay. The apoptotic nurse cell remnants are phagocytosed by the neighboring follicle cells at the end of oogenesis during stages 13 and 14. In the second apoptotic pattern, which occurs sporadically during stages 7 and 8, the nurse cells degenerate and are phagocytosed by the follicular epithelium that contains apoptotic cell bodies. The data presented herein, compared to previous reported results in Drosophila melanogaster and Dacus oleae (Nezis et al., 2000, 2001), strongly suggest that nurse cell apoptosis is a developmentally regulated and phylogenetically conserved mechanism in higher Dipteran. They also suggest that, the sporadic apoptotic pattern consists of a possible protective mechanism throughout oogenesis when damaged or abnormal egg chambers, are eliminated before they reach maturity.
Abstract: In the present study, we demonstrate the apoptotic events of the ovarian follicle cells during the late stages of oogenesis in Drosophila melanogaster. Follicle cell morphology appears normal from stage 10 up to stage 14, exhibiting a euchromatic nucleus and a well-organized cytoplasm. First signs of apoptosis appear at the anterior pole of the egg chamber at stage 14A. They are characterized by loss of microvilli at the apical cell membrane, alterations in nuclear morphology, such as chromatin condensation and convolution of the nuclear membrane, and also by condensation and vacuolization of the cytoplasm. During the following stage 14B, the follicle cell nuclei contain fragmented DNA as is demonstrated by acridine orange staining and TUNEL (TdT-mediated dUTP nick end-labeling) assay. Finally, the apoptotic follicle cells seem to detach from the eggshell when the mature egg chamber exits the ovariole. The detached follicle cells exhibit condensed nuclear chromatin, a disorganized cytoplasm with crowded organelles and are surrounded by epithelial cells. The above results seem to be associated with the abundant phagocytosis that we observed at the entry of the lateral oviducts, where the epithelial cells contain apoptotic cell bodies. Additionally, we tested the effect of etoposide treatment in the follicular epithelium and found that it induces apoptosis in a stage- and site-specific manner. These observations suggest a possible method of absorption of the apoptotic follicle cells that prevents the blockage of the ovarioles and helps the regular production of mature eggs.
Abstract: We report, for the first time, an unusual case of congenital anaemia with the clinical diagnosis of haemoglobin H disease complicated by morphological features at the light and electron microscopy level very similar to those of CDA-I. The red cell indices and the globin chain biosynthetic ratio were not characteristic of the defective haemoglobin genotype. The haematological, clinical and morphological data strongly suggest the novel coexistence of the two defects in a patient. The disease is characterised by a unique dyserythropoietic phenotype of diagnostic importance, which possibly brings new data regarding the reciprocal interaction between the two diseases, especially concerning a specific abnormality in globin chain synthesis in CDA-I, as previously suggested.
Abstract: In the present study, we demonstrate the actin cytoskeleton reorganization during nurse cells apoptosis of the olive fruit fly Dacus oleae. At the developmental stage 9A of oogenesis, the actin microfilaments are assembled in numerous ring canals and subcortically support all the nurse cells, as is shown by phalloidin-FITC staining. During the following stages, 9B and 10A, this structural pattern remains the same. The developmental stage 10B is characterized by actin microfilament rearrangement and formation of actin cables that are symmetrically organized around the nurse cell nuclei. At stage 11, when the dumping process begins, these actin cables seem to retain each nurse cell nucleus in the cell center, away from blocking the ring canals. The early stage 12 is characterized by an asynchronous nurse cell nuclear chromatin condensation, while at late stage 12 the actin cables become very thick, as adjacent ones overlap one another and traverse the disorganized apoptotic nurse cell nuclei that already have fragmented DNA, as is demonstrated by acridine orange staining and TUNEL assay. Finally, during stage 13, the apoptotic nuclear remnants are phagocytosed by the neighboring follicle cells. The data presented herein compared to previous reported results in Drosophila [Nezis et al., 2000: Eur J Cell Biol 79:610-620], demonstrate that actin cytoskeleton reorganization during nurse cell apoptosis is a developmentally regulated physiological mechanism, phylogenetically conserved in higher Dipteran.
Abstract: Conventional and freeze-fracture electron microscopy, immuno-electron microscopy of ovarian cryosections and confocal immunofluorescence were used to analyze the ovarian distribution of the major protein classes being secreted by the follicle cells during the vitellogenic and choriogenic stages of Drosophila oogenesis. Our results clearly demonstrated that at vitellogenic stages the follicle cells co-secrete constitutively vitelline membrane and yolk proteins that are either sorted into distinct secretory vesicles or they are segregated in different parts of bipartite vesicles by differential condensation. Following their exocytosis only the vitelline membrane proteins are incorporated into the forming vitelline membrane. The yolk proteins (along with their hemolymph circulating counterparts) diffuse through gaps amongst the incomplete vitelline membrane and are internalized through endocytosis by the oocyte where they are finally stored into modified lysosomes referred to as alpha-yolk granules. The unexpected immunolocalization of vitelline membrane antigens in the associated body of the alpha-yolk granules may indicate that this structure is a transient repository for the proteins being internalized into the oocyte along with the yolk proteins. In the early choriogenic follicle cells the vitelline membrane and early chorion proteins were found to be co-secreted and to be evenly intermixed into the same secretory vesicles. These findings illuminate new details concerning the follicle cells secretory and oocyte endocytic pathways and provide for the first time evidence for condensation-mediated sorting of constitutively secreted proteins in Drosophila.
Abstract: In the present study we demonstrate the existence of two apoptotic patterns in Drosophila nurse cells during oogenesis. One is developmentally regulated and normally occurs at stage 12 and the other is stage-specific and is sporadically observed at stages 7 and 8 of abnormally developed follicles. The apoptotic manifestation of the first pattern begins at stage 11 and is marked by a perinuclear rearrangement of the actin cytoskeleton and the development of extensive lobes and engulfments of the nurse cell nuclei located proximal to the oocyte. Consequently, at late stage 12 (12C), half of the nurse cell nuclei exhibit condensed chromatin, while at late stage 13 all the nuclei have fragmented DNA, as it is clearly shown by TUNEL assay. Finally, the apoptotic vesicles that are formed during stage 13, are phagocytosed by the neighboring follicle cells and at stage 14 the nurse cell nuclear remnants can be easily detected within the adjacent follicle cell phagosomes. In the second sporadic apoptotic pattern, all the nurse cell nuclei are highly condensed with fragmented DNA, accompanied by a completely disorganized actin cytoskeleton. When we induced apoptosis in Drosophila follicles through an etoposide and staurosporine in vitro treatment, we observed a similar pattern of stage-specific cell death at stages 7 and 8. These observations suggest a possible protective mechanism throughout Drosophila oogenesis that results in apoptosis of abnormal, damaged or spontaneously mutated follicles before they reach maturity.
Abstract: The innermost chorionic layer (ICL) in eggshells of Drosophila melanogaster is a naturally occurring patchwork of thin three-dimensional crystalline plates located between the inner endochorion and the vitelline envelope. The mass-per-unit area of the ICL has been measured from scanning transmission electron microscope images of isolated unstained material and it was possible to distinguish up to four layers with the majority of the crystalline sheets being one to three layers thick. Taking into account the unit cell areas for the different crystals, we have estimated the mean ICL subunit sizes to be 36 kDa for Drosophila melanogaster, 35 kDa for Drosophila auraria, and 33 kDa for Drosophila teissieri. The results suggest that the three different Drosophilidae species have very similar average subunit masses.
Abstract: Synthesis and morphogenesis of the innermost chorionic layer (ICL) was investigated by conventional EM methods, freeze-fracturing, tissue culture in Robb's medium, and EM autoradiography. Both autoradiography and fine structure results have shown that ICL-components are secreted prior to other chorion proteins. Their secretion starts on stage 12a but the first layer of ICL molecules is visible at stage 12b. Its thickness is gradually increased during the next stages, taking first, a bilaminar form along with the inner endochorion. Later, at the end of choriogenesis, ICL is detached from the endochorion and takes its final thickness and configuration, consisting of a 3-dimensional crystal, about 40 nm thick. The isolated ICL in conditions of air water interface is a monolayer crystal 10 nm thick. Studies on chorion mutants showed that the amount of protein secreted by the follicle cells is independent to the process of crystallisation. These data show how a proteinaceous extracellular substance is gradually assembled to form a 3-D crystal and how it can be organised to perform functions such as the physiological resistance of the insect eggs against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. These functions are performed by ICL in conjunction with the underlying wax layer.
Abstract: Utilizing freeze-fracturing and conventional electron microscopy methods, we have studied the details of morphogenesis and construction of the wax layer envelope from Oregon R and mutants of Drosophila melanogaster eggs during oogenesis. The wax layer is synthesized and secreted by the follicular cells in the form of lipid vesicles during stage 10b. During secretion (stages 10b, 11 and 12) the lipid vesicles are accumulated on the vitelline membrane surface and become flat. At the late stages of choriogenesis (stages 13, 14) the lipid vesicles are compressed tightly between the vitelline membrane and the other already constructed eggshell layers, so the wax layer becomes very thin and is hardly seen in cross-fractured views.
Abstract: A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.
Abstract: Utilizing freeze-fracturing conventional electron microscopy and scanning electron microscopy methods, a wax layer was identified, sealing the oocyte of Drosophila melanogaster. In mature egg-shells wax forms a hydrophobic layer surrounding the oocyte and lying between, and in very close contact with the vitelline membrane (interiorly) and the crystalline intermediate chorionic layer (exteriorly). In cross-fractured views it is less than 50 A thick whereas in longitudinal fracturing it reveals smooth fracture faces of a multilayered material in the form of hydrophobic areas or plaques (0.5-1 microns in diameter) which are partially overlapping and highly compressed between the vitelline membrane and the innermost chorionic layer. The evidence for this layer being a wax are the facts that a) it is not preserved in conventional fat-extracting electron microscopy methods, b) it directs laterally the fracture planes during freeze-fracturing and reveals smooth fracture faces. Analysis of the structural features of wax in mature egg-shell in various species of Drosophilidae have shown that the wax layer exhibits indistinguishable (among the species) hydrophobic plaques, which have the same size and thickness with Drosophila melanogaster. These data provide structural evidence explaining the physiological resistance of the insect eggs studied, against water loss or water uptake, whenever they are laid on substrates with extreme environmental conditions. In addition, the data demonstrate how an extracellular substance can be organized to perform that function.
