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H Schatten

Publications and source records attributed to H Schatten.

At least 37 records · Page 2Linked to original sources
PubMed1999

Centrosome and microtubule instability in aging Drosophila cells.

Several cytoskeletal changes are associated with aging which includes alterations in muscle structure leading to muscular atrophy, and weakening of the microtubule network which affects cellular secretion and maintenance of cell shape. Weakening of the microtubule network during meiosis in aging oocytes can result in aneuploidy or trisomic zygotes with increasing maternal age. Imbalances of cytoskeletal organization can lead to disease such as Alzheimer's, muscular disorders, and cancer. Because many cytoskeletal diseases are related to age we investigated the effects of aging on microtubule organization in cell cultures of the Drosophila cell model system (Schneider S-1 and Kc23 cell lines). This cell model is increasingly being used as an alternative system to mammalian cell cultures. Drosophila cells are amenable to genetic manipulations and can be used to identify and manipulate genes which are involved in the aging processes. Immunofluorescence, scanning, and transmission electron microscopy were employed for the analysis of microtubule organizing centers (centrosomes) and microtubules at various times after subculturing cells in fresh medium. Our results reveal that centrosomes and the microtubule network becomes significantly affected in aging cells after 5 days of subculture. At 5-14 days of subculture, 1% abnormal out of 3% mitoses were noted which were clearly distinguishable from freshly subcultured control cells in which 3% of cells undergo normal mitosis with bipolar configurations. Microtubules are also affected in the midbody during cell division. The midbody in aging cells becomes up to 10 times longer when compared with midbodies in freshly subcultured cells. During interphase, microtubules are often disrupted and disorganized, which may indicate improper function related to transport of cell organelles along microtubules. These results are likely to help explain some cytoskeletal disorders and diseases related to aging.

Animals↗
PubMed1999

Activation of Sp1 and its functional co-operation with serum amyloid A-activating sequence binding factor in synoviocyte cells trigger synergistic action of interleukin-1 and interleukin-6 in serum amyloid A gene expression.

The serum amyloid A (SAA) protein has been implicated in the progression and pathogenesis of rheumatoid arthritis through induction of collagenase activity in synovial fibroblast cells that line the joint tissues. We demonstrate that SAA is synergistically induced in synovial cells by interleukin (IL)-1 and IL-6 that are present at significantly high level in the synovial fluid of arthritis patients. These cytokines induced phenotypic changes in synovial cells, promoting protrusion and increased cellular contact. Induction of SAA under this condition is mediated by promoter elements located between -254 and -226, which contains binding sites for transcription factors Sp1 and SAA activating sequence binding factor (SAF). Mutation of these sequences abolishes SAA promoter response to IL-1 and IL-6. The role of Sp1 in SAA induction was demonstrated by increased DNA binding activity, phosphorylation, and increased protein content of Sp1 during cytokine treatment. Sp1 interacts with the SAA promoter in association with SAF as an SAF. Sp1 heteromeric complex. Furthermore, using a phosphatase inhibitor, we demonstrated increased transactivation potential of both Sp1 and SAF as a consequence of a phosphorylation event. These results provide first evidence for cytokine-mediated activation of Sp1 in synovial fibroblast cells and its participation in regulating SAA expression by acting in conjunction with SAF.

Animals↗
PubMed1999

Effects of spaceflight conditions on fertilization and embryogenesis in the sea urchin Lytechinus pictus.

Calcium loss and muscle atrophy are two of the main metabolic changes experienced by astronauts and crew members during exposure to microgravity in space. Calcium and cytoskeletal events were investigated within sea urchin embryos which were cultured in space under both microgravity and 1 g conditions. Embryos were fixed at time-points ranging from 3 h to 8 days after fertilization. Investigative emphasis was placed upon: (1) sperm-induced calcium-dependent exocytosis and cortical granule secretion, (2) membrane fusion of cortical granule and plasma membranes; (3) microfilament polymerization and microvilli elongation; and (5) embryonic development into morula, blastula, gastrula, and pluteus stages. For embryos cultured under microgravity conditions, the processes of cortical granule discharge, fusion of cortical granule membranes with the plasma membrane, elongation of microvilli and elevation of the fertilization coat were reduced in comparison with embryos cultured at 1 g in space and under normal conditions on Earth. Also, 4% of all cells undergoing division in microgravity showed abnormalities in the centrosome-centriole complex. These abnormalities were not observed within the 1 g flight and ground control specimens, indicating that significant alterations in sea urchin development processes occur under microgravity conditions.

Animals↗
PubMed1999

Role of the MAPK cascade in mammalian germ cells.

The mitogen-activated protein kinase (MAPK) cascade is one of the most important of the intracellular signaling pathways that play a crucial role in cell proliferation, cell differentiation and cell cycle regulation. Since the first report in 1993 of MAPK's involvement in the functional regulation of mammalian oocytes, much work has been done on the role of the MAPK cascade in germ cells in different species of mammals. This review describes the possible involvement of the MOS/MEK/MAPK/RSK cascade in spermatogenesis, sperm function, oocyte meiotic re-initiation, spindle assembly, metaphase II arrest, pronuclear formation and the entry of first mitosis, as well as the cross-talk of this cascade to maturation-promoting factor (MPF) and other signal molecules in mammals.

Animals↗
PubMed1999

Androgen-induced oxidative stress in human LNCaP prostate cancer cells is associated with multiple mitochondrial modifications.

We investigated the role of androgen-induced oxidative stress in prostate cancer using the androgen-responsive LNCaP human prostate cancer cell line exposed to a 1-nM concentration of the synthetic androgen R1881 (which correlates with serum androgen levels). Such exposure, which decreases growth rate and increases oxidative stress in LNCaP cells, induced statistically significant mitochondrial changes. A 40% increase in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, indicative of mitochondrial dehydrogenase activity, occurred 24 hr after androgen treatment. This change preceded 50-110% increases, 40-96 hr after R1881 exposure, in levels of cellular peroxides and hydroxyl radicals as measured by 2'7'-dicholorofluorescin diacetate (DCF) fluorescence. On the basis of electron microscopy measurements, R1881 treatment increased the area fraction of mitochondria per cell by approximately 100% at 72 hr. In agreement, mitochondrial mass at 96 hr, evaluated by the fluorescent dye nonyl acridine orange (NAO), was 80% higher in treated cells. R1881 exposure for 24 hr lowered the activities of electron transport system (ETS) complexes, I, II, and IV by 17-27% and ATP levels by 50%. The ETS inhibitors, rotenone and antimycin A, lowered androgen-induced DCF fluorescence readings to control levels thereby suggesting ETS involvement in androgen-induced oxidant production. Addition of alpha-tocopherol succinate abrogated R1881-induced elevations in MTT reduction. In sum, androgens may, directly or indirectly, contribute to oxidative stress in LNCaP cells by regulating mitochondrial number, activity, and oxidant production by mechanisms that are, at least in part, sensitive to an antioxidant.

Antimycin A↗
PubMed1999

Sea urchin fertilization during a KC-135 parabolic flight.

For long-term exposure to space it is crucial to understand the underlying mechanisms for altered physiological functions. We have chosen the sea urchin system to study the effects of microgravity on various cellular processes visible during fertilization and subsequent development. We report here on experiments performed on NASA's KC-135 during parabolic flight trajectories to validate procedures to be implemented as part of the first Aquatic Research Facility Space Shuttle experiment on STS-77.

Animals↗
PubMed1999

Utilization of the aquatic research facility and fertilization syringe unit to study sea urchin development in space.

Methods were developed for the investigation of the effects of microgravity on early development in sea urchins within the Canadian Space Agency's Aquatic Research Facility (ARF). The ARF payload provided light, temperature control, automated fixation capability, and a 1 G on-orbit centrifuge control. Eggs and embryos of either the sea urchin species Lytechinus pictus or Strongylocentrotus purpuratus were loaded into Standard Container Assemblies (SCAs) which comprised the experimental aquaria (33 mL volume) contained within the ARF. A newly developed Fertilization Syringe Unit (FSU) was used to achieve "in-flight" fertilization capability. Fixative solutions were preloaded into fixation blocks maintained adjacent to the SCAs and injected at pre-selected time points, resulting in final (diluted) concentrations of either 0.5% or 2% glutaraldehyde (depending upon embryonic stage). Light, scanning, and transmission electron microscopy determined that all desired embryonic and cell division stages (16-cell stage, blastula, gastrula, and pluteus) were preserved using the experimental protocols and fixation capability provided by the ARF/FSU system.

Animals↗
PubMed1998

Chloral hydrate alters the organization of the ciliary basal apparatus and cell organelles in sea urchin embryos.

The mitotic inhibitor, chloral hydrate, induces ciliary loss in the early embryo phase of Lytechinus pictus. It causes a breakdown of cilia at the junction of the cilium and the basal body known as the basal plate. This leaves the plasma membrane temporarily unsealed. The basal apparatus accessory structures, consisting of the basal body, basal foot, basal foot cap, striated side arm, and striated rootlet, are either misaligned or disintegrated by treatment with chloral hydrate. Furthermore, microtubules which are associated with the basal apparatus are disassembled. Mitochondria accumulate at the base of cilia - underneath the plasma membrane - and show alterations in their structural organization. The accumulation of mitochondria is observed in 40% of all electron micrograph sections while 60% show the areas mostly devoid of mitochondria. The microvilli surrounding a cilium and striated rootlet remain intact in the presence of chloral hydrate. These results suggest that deciliation in early sea urchin embryos by chloral hydrate is caused by combined effects on the ciliary membrane and on microtubules in the cilia. Furthermore, it is suggested that chloral hydrate can serve as a tool to explore the cytoskeletal mechanisms that are involved in cilia motility in the developing sea urchin embryo.

Animals↗
PubMed1998

The cytoskeleton of Drosophila-derived Schneider line-1 and Kc23 cells undergoes significant changes during long-term culture.

Insect cell cultures derived from Drosophila melanogaster are increasingly being used as an alternative system to mammalian cell cultures, as they are amenable to genetic manipulation. Although Drosophila cells are an excellent tool for the study of genes and expression of proteins, culture conditions have to be considered in the interpretation of biochemical results. Our studies indicate that significant differences occur in cytoskeletal structure during the long-term culture of the Drosophila-derived cell lines Schneider Line-1 (S1) and Kc23. Scanning, transmission-electron, and immunofluorescence microscopy studies reveal that microfilaments, microtubules, and centrosomes become increasingly different during the culture of these cells from 24 h to 7-14 days. Significant cytoskeletal changes are observed at the cell surface where actin polymerizes into microfilaments, during the elongation of long microvilli. Additionally, long protrusions develop from the cell surface; these protrusions are microtubule-based and establish contact with neighboring cells. In contrast, the microtubule network in the interior of the cells becomes disrupted after four days of culture, resulting in altered transport of mitochondria. Microtubules and centrosomes are also affected in a small percent of cells during cell division, indicating an instability of centrosomes. Thus, the cytoskeletal network of microfilaments, microtubules, and centrosomes is affected in Drosophila cells during long-term culture. This implies that gene regulation and post-translational modifications are probably different under different culture conditions.

Actin Cytoskeleton↗
PubMed1998

Centrosome structure and function is altered by chloral hydrate and diazepam during the first reproductive cell cycles in sea urchin eggs.

This paper explores the mode of action of the tranquillizers chloral hydrate and diazepam during fertilization and mitosis of the first reproductive cell cycles in sea urchin eggs. Most striking effects of these drugs are the alteration of centrosomal material and the abnormal microtubule configurations during exposure and after recovery from the drugs. This finding is utilized to study the mechanisms of centrosome compaction and decompaction and the dynamic configurational changes of centrosomal material and its interactions with microtubules. When 0.1% chloral hydrate or 350-750 microM diazepam is applied at specific phases during the first cell cycle of sea urchin eggs, expanded centrosomal material compacts at distinct regions and super-compacts into dense spheres while microtubules disassemble. When eggs are treated before pronuclear fusion, centrosomal material aggregates around each of the two pronuclei while microtubules disappear. Upon recovery, atypical asters oftentimes with multiple foci are formed from centrosomal material surrounding the pronuclei which indicates that the drugs have affected centrosomal material and prevent it from functioning normally. Electron microscopy and immunofluorescence studies with antibodies that routinely stain centrosomes in sea urchin eggs (4D2; and Ah-6) depict centrosomal material that is altered when compared to control cells. This centrosomal material is not able to reform normal microtubule patterns upon recovery but will form multiple asters around the two pronuclei. When cells are treated with 0.1% chloral hydrate or 350-750 microM diazepam during mitosis, the bipolar centrosomal material becomes compacted and aggregates into multiple dense spheres while spindle and polar microtubules disassemble. With increased incubation time, the smaller dense centrosome particles aggregate into bigger and fewer spheres. Upon recovery, unusual irregular microtubule configurations are formed from centrosomes that have lost their ability to reform normal mitotic figures. These results indicate that chloral hydrate and diazepam affect centrosome structure which results in the inability to reform normal microtubule formations and causes abnormal fertilization and mitosis.

Animals↗
PubMed1996

Cold-treated centrosome: isolation of centrosomes from mitotic sea urchin eggs, production of an anticentrosomal antibody, and novel ultrastructural imaging.

A novel isolation of centrosomes is described and it was used to both generate a centrosome-specific monoclonal antibody and to image with high-resolution low-voltage scanning electron microscopy the surface details of the isolated centrosome. At first mitotic prometaphase, sea urchin zygotes are chilled on ice overnight. While most of the microtubules disassemble, the mitotic centrosomes collapse into aggregated masses. These centrosomes have been isolated, and used to generate a monoclonal antibody, designated 4D2, which is reactive with interphase and mitotic centrosomes. 4D2 staining of centrosomes is similar, but not identical, to that of other centrosomal antibodies like Ah6 and 5051. Centrosomal material is detected as a compact sphere after cold treatment; upon recovery the sphere expands and undergoes the shape changes previously described [Mazia et al., 1987: J. Cell Biol. 105:206a] to eventually reorganize a normal mitotic apparatus.

Animals↗
PubMed1994

Dithiothreitol prevents membrane fusion but not centrosome or microtubule organization during the first cell cycles in sea urchins.

Dithiothreitol (DTT), a disulfide reducing agent, inhibits the fusion of male and female pronuclei within the activated cytoplasm of sea urchin eggs. The migrations of the pronuclei are not affected by DTT, indicating that microtubule function is not impaired. Centrosomal antigens are detected in the sperm aster and in all subsequent microtubule-based configurations. Nuclear membranes never fuse and the chromatin of male and female pronuclei never mix in the DTT-treated cells. During prophase, when nuclear envelopes break down to undergo mitosis, both sets of chromosomes undergo condensation cycles independent from each other. Both pronuclei initially stain for centrosomal material and surrounding microtubules. With time, the female's centrosomal material as well as the microtubules disappear while the male forms a bipolar spindle. Interestingly, one pole of the paternal mitotic apparatus communicates with the separate maternal chromatin, forming a half spindle which moves the egg-derived chromatin towards its pole. At the time for cell division, the individual karyomeres are not able to fuse their nuclear membranes to reconstitute the blastomere nuclei. When DTT is applied at prometaphase of the first cell cycle, the chromosome cycle continues until next metaphase. Centrosomes also continue their cycle and undergo somewhat atypical splitting during the time for second telophase. Division furrows are initiated but aborted. These results support the hypothesis that disulfide groups are required for membrane fusion of the pronuclei, for membrane fusion of the karyomeres, and for the completion of the division furrow to achieve successful cell division.

Animals↗
PubMed1992

Activation of maternal centrosomes in unfertilized sea urchin eggs.

Centrosomes are undetectable in unfertilized sea urchin eggs, and normally the sperm introduces the cell's microtubule-organizing center (MTOC) at fertilization. However, artificial activation or parthenogenesis triggers microtubule assembly in the unfertilized egg, and this study explores the reappearance and behavior of the maternal centrosome. During activation with A23187 or ammonia, microtubules appear first at the cortex; centrosomal antigen is detected diffusely throughout the entire cytoplasm. Later, the centrosome becomes more distinct and organizes a radial microtubule shell, and eventually a compact centrosome at the egg center organizes a monaster. In these activated eggs, centrosomes undergo cycles of compaction and decompaction in synchrony with the chromatin, which also undergoes cycles of condensation and decondensation. Parthenogenetic activation with heavy water (50% D2O) or the microtubule-stabilizing drug taxol (10 microM) induces numerous centrosomal foci in the unfertilized sea urchin egg. Within 15 min after incubation in D2O, numerous fine centrosomal foci are detected, and they organize a connected network of numerous asters which fill the entire egg. Taxol induces over 100 centrosomal foci by 15 min after treatment, which organize a corresponding number of asters. The centrosomal material in either D2O- or taxol-treated eggs aggregates with time to form fewer but denser foci, resulting in fewer and larger asters. Fertilization of eggs pretreated with either D2O or taxol shows that the paternal centrosome is dominant over the maternal centrosome. The centrosomal material gradually becomes associated with the enlarged sperm aster. These experiments demonstrate that maternal centrosomal material is present in the unfertilized egg, likely as dispersed undetectable material, which can be activated without paternal contributions. At fertilization, paternal centrosomes become dominant over the maternal centrosomal material.

Ammonia↗
PubMed1992

Microgravity effects on sea urchin fertilization and development.

Gravity has been a pervasive influence on all living systems and there is convincing evidence to suggest that it alters fertilization and embryogenesis in several developmental systems. Notwithstanding the global importance of gravity on development, it has only been recently possible to begin to design experiments which might directly investigate the specific effects of this vector. The goal of this research program is to explore and understand the effects of gravity on fertilization and early development using sea urchins as a model system. Sea urchin development has several advantages for this project including the feasibility of maintaining and manipulating these cells during spaceflight, the high percentage of normal fertilization and early development, and the abundant knowledge about molecular, biochemical, and cellular events during embryogenesis which permits detailed insights into the mechanism by which gravity might interfere with development. Furthermore, skeletal calcium is deposited into the embryonic spicules within a day of fertilization permitting studies of the effects of gravity on bone calcium deposition.

Animals↗
PubMed1991

Maternal inheritance of centrosomes in mammals? Studies on parthenogenesis and polyspermy in mice.

The centrosome, the microtubule-organizing center of the cell, is introduced typically by the sperm at fertilization. In some mammals, however, this paternal pattern of inheritance appears to be violated. The hypothesis that the centrosome is maternally inherited was tested during parthenogenesis, polyspermy, and polygyny as well as after recovery from microtubule inhibition at first mitosis. During parthenogenesis the paternal contribution was absent, and in polyspermy the paternal contribution was multiplied. Haploid and diploid parthenogenotes as well as polyspermic and digynic fertilized eggs each segregated their centrosomes to organize a bipolar mitotic apparatus. Oocytes recovering from a nocodazole block formed two normal bipolar mitotic apparatus; the paternal chromosomes aligned at one spindle equator, while the maternal chromosomes were found at the other. These results show that the centrosome is maternally inherited from cytoplasmic sites in the mouse. The evolutionary switch from paternal to maternal inheritance in mammals might be related to the additional dangers that parthenogenesis represents: a threat to the life of the mother as well as to the life of the fetus.

Animals↗
PubMed1990

T-1, a mitotic arrester, alters centrosome configurations in fertilized sea urchin eggs.

T-1 induces modifications in the shape of the centrosome at division in fertilized eggs of the North American sea urchin, Lytechinus pictus. Phase contrast microscopy observations of mitotic apparatus isolated from T-1-treated (1.7-8.5 microM) eggs at first division shows that the centrosomes already begin to spread or to separate by prophase and that the mitotic spindle is barrel-shaped. When eggs are fertilized with sperm that have been preteated with T-1, the centrosomes become flattened; the spindles are of normal length. Immunofluorescence microscopy using an anti-centrosomal monoclonal antibody reveals that T-1 modifies the structure of the centrosome so that barrel-shaped spindles with broad centrosomes are observed at metaphase, rather than the expected focused poles and fusiform spindle. Higher concentrations of T-1 induce fragmentation of centrosomes, causing abnormal accumulation of microtubules in polar regions. These results indicate that T-1 directly alters centrosomal configuration from a compact structure to a flattened or a spread structure. T-1 can be classified as a new category of mitotic drugs that may prove valuable in dissecting the molecular nature of centrosomes.

Animals↗