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C S Navara

Publications and source records attributed to C S Navara.

18 recordsLinked to original sources
PubMed2001

Vanadocenes as potent anti-proliferative agents disrupting mitotic spindle formation in cancer cells.

We present experimental data which establish the organometallic compounds vanadocene dichloride (VDC) and vanadocene acetylacetonate (VDacac) as potent anti-proliferative agents. We first examined the effects of VDC and VDacac on the rapid embryonic cell division and development of Zebrafish. Both compounds were capable of causing cell division block at the 8-16 cell stage of embryonic development followed by total cell fusion and developmental arrest. We next examined the effect of VDC and VDacac on proliferation of human breast cancer and glioblastoma cell lines using MTT assays. VDC inhibited the proliferation of the breast cancer cell line BT-20 as well as the glioblastoma cell line U373 in a concentration-dependent fashion with IC50 values of 11.0, 14.9 and 18.6 microM, respectively. VDacac inhibited cellular proliferation with IC50 values of 9.1, 26.9 and 35.5 microM, respectively. Whereas in vehicle-treated control cancer cells mitotic spindles were organized as a bipolar microtubule array and the DNA was organized on a metaphase plate, vanadocene-treated cancer cells had aberrant monopolar mitotic structures where microtubules were detected only on one side of the chromosomes and the chromosomes were arranged in a circular pattern. In contrast to control cells which showed a single focus of gamma-tubulin at each pole of the bipolar mitotic spindle, VDC- or VDacac-treated cells had two foci of gamma-tubulin on the same side of the chromosomes resulting in a broad centrosome at one pole. All monopolar spindles examined had two foci of gamma-tubulin labeling consistent with a mechanism in which the centrosomes duplicate but do not separate properly to form a bipolar spindle. These results provide unprecedented evidence that organometallic compounds can block cell division in human cancer cells by disrupting bipolar spindle formation. In accordance with these results vanadocene treatment caused an arrest at the G2/M phase of the cell cycle. This unique mechanism of anti-mitotic function warrants further development of vanadocene complexes as anti-cancer drugs.

Animals↗
PubMed2001

Spongistatins as tubulin targeting agents.

Recently identified novel agents that disrupt tubulin polymerization include synthetic spiroketal pyrans (SPIKET) targeting the spongistatin binding site of b-tubulin. These agents exhibit anticancer activity by disrupting normal mitotic spindle assembly and cell division as well as inducing apoptosis. At nanomolar concentrations, the SPIKET compound SPIKET-P caused tubulin depolymerization in cell-free turbidity assays and exhibited potent cytotoxic activity against cancer cells as evidenced by destruction of microtubule organization, and prevention of mitotic spindle formation in human breast cancer cells. SPIKET compounds represent a new class of tubulin targeting agents that show promise as anti-cancer drugs.

Animals↗
PubMed2001

Structure-based design of novel anticancer agents.

Recently identified agents that interact with cytoskeletal elements such as tubulin include synthetic spiroketal pyrans (SPIKET) and monotetrahydrofuran compounds (COBRA compounds). SPIKET compounds target the spongistatin binding site of beta-tubulin and COBRA compounds target a unique binding cavity on alpha-tubulin. At nanomolar concentrations, the SPIKET compound SPIKET-P causes tubulin depolymerization and exhibits potent cytotoxic activity against cancer cells. COBRA-1 inhibits GTP-induced tubulin polymerization. Treatment of human breast cancer and brain tumor cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Other studies have identified some promising protein tyrosine kinase inhibitors as anti-cancer agents. These include EGFR inhibitors such as the quinazoline derivative WHI-P97 and the leflunomide metabolite analog LFM-A12. Both LFM-A12 and WHI-P97 inhibit the in vitro invasiveness of EGFR positive human breast cancer cells at micromolar concentrations and induce apoptotic cell death. Dimethoxyquinazoline compounds WHI-P131 and WHI-P154 inhibit tyrosine kinase JAK3 in leukemia cells. Of particular interest is WHI-P131, which inhibits JAK3 but not JAK1, JAK2, SYK, BTK, LYN, or IRK at concentrations as high as 350 microM. Studies of BTK inhibitors showed that the leflunomide metabolite analog LFM-A13 inhibited BTK in leukemia and lymphoma cells. Consistent with the anti-apoptotic function of BTK, treatment of leukemic cells with LFM-A13 enhanced their sensitivity to chemotherapy-induced apoptosis.

Animals↗
PubMed2000

COBRA-1, a rationally-designed epoxy-THF containing compound with potent tubulin depolymerizing activity as a novel anticancer agent.

A novel mono-THF containing synthetic anticancer drug, COBRA-1, was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of alpha-tubulin. COBRA-1 inhibited GTP-induced tubulin polymerization in cell-free tubulin turbidity assays. Treatment of human breast cancer and brain tumor (glioblastoma) cells with COBRA-1 caused destruction of microtubule organization and apoptosis. Like other microtubule-interfering agents, COBRA-1 activated the proapoptotic c-Jun N-terminal kinase (JNK) signal transduction pathway, as evidenced by rapid induction of c-jun expression.

Antineoplastic Agents↗
PubMed2000

A rationally designed anticancer drug targeting a unique binding cavity of tubulin.

A novel mono-THF containing synthetic anticancer drug (WHI-261) was designed for targeting a previously unrecognized unique narrow binding cavity on the surface of tubulin. The anti-cancer activity of WHI-261 was confirmed using MTT assays. The structure-based design, synthesis, and biological activity of WHI-261 are reported.

Antineoplastic Agents↗
PubMed2000

SPIKET and COBRA compounds as novel tubulin modulators with potent anticancer activity.

Agents that either promote or inhibit tubulin polymerization exhibit anticancer activity by disrupting normal mitotic spindle assembly and cell division as well as inducing apoptosis. Recently identified novel agents that target tubulin include synthetic spiroketal pyrans (SPIKET), targeting the spongistatin binding site of beta-tubulin, and COBRA compounds, targeting a unique binding cavity on alpha-tubulin. At nanomolar concentrations, the SPIKET compound SPIKET-P caused tubulin depolymerization in cell-free turbidity assays and exhibited potent cytotoxic activity against cancer cells as evidenced by destruction of microtubule organization, and prevention of mitotic spindle formation in human breast cancer cells. Molecular modeling studies predicted a high-affinity interaction of the first COBRA compounds, COBRA-0 and COBRA-1, with a unique hydrophobic binding site on alpha-tubulin located between the GTP/GDP binding site and the M-loop. Further studies showed that COBRA-1 inhibited GTP-induced tubulin polymerization in cell-free tubulin turbidity assays. Treatment of human breast cancer and brain tumor (glioblastoma) cells with COBRA-1 caused destruction of microtubule organization and apoptosis. COBRA-1 activated the pro-apoptotic c-Jun N-terminal kinase (JNK) signal transduction pathway. COBRA and SPIKET compounds represent two new classes of tubulin targeting agents that show promise as anticancer drugs.

Animals↗
PubMed1999

Biparental inheritance of gamma-tubulin during human fertilization: molecular reconstitution of functional zygotic centrosomes in inseminated human oocytes and in cell-free extracts nucleated by human sperm.

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents gamma-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. gamma-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal gamma-tubulin. Recruitment of maternal gamma-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm "priming" induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Animals↗
PubMed1997

Mitochondrial sheath movement and detachment in mammalian, but not nonmammalian, sperm induced by disulfide bond reduction.

The successful completion of the fertilization process requires the properly choreographed unsheathing of the tightly packaged sperm once it has been fully incorporated into the egg's cytoplasm. The nuclear and accessory structures of mammalian sperm become stabilized by disulfide bonds (S-S) during epididymal maturation. This stabilization is reversed during fertilization by the reduction of S-S cross-linking, but little is known about the effect of S-S reduction on individual disulfide-hardened structures such as the sperm's connecting piece, fibrous sheath, and mitochondria. Here, we demonstrate the action of the S-S-reducing environment on the mitochondrial sheath of mammalian sperm, visualized by the vital fluorescent probe MitoTracker and by electron microscopy. In both human and bull sperm, mitochondria form a compact helix (mitochondrial sheath) wrapped around the midpiece and connecting piece that can be fluorescently labelled by a short incubation with 100 nM MitoTracker. Exposure of bull sperm to 0.1-10 mM dithiothreitol (DTT; a disulfide bond-reducing agent) induced a time and dose-dependent sliding of the mitochondrial sheath down the axoneme, accompanied by the excision of the sperm tail and decondensation of the sperm nucleus. Increasing the concentration of DTT to 100 mM accelerated mitochondrial movement, causing a completed stripping of sperm mitochondria and partial disassembly of the connecting piece. Likewise, human sperm responded to DTT treatment by the sliding or removal of the mitochondrial sheath and decondensation of the sperm chromatin. These events were not observed in the sperm of lower vertebrates and invertebrates (Xenopus laevis and Lytechinus pictus, respectively) exposed to an excess of DTT. Thus the sensitivity of sperm mitochondria to the S-S reducing environment seems to be an exclusive feature of mammalian sperm. The movement of sperm mitochondria induced by S-S reduction may be an initial critical step in the disassembly of the mammalian sperm tail during fertilization.

Amphibians↗
PubMed1997

The implications of a paternally derived centrosome during human fertilization: consequences for reproduction and the treatment of male factor infertility.

PROBLEM: Successful fertilization in humans follows a complex series of events, including the completion of meiotic maturation of the oocyte with the extrusion of the second polar body, the decondensation of the sperm nucleus and the maternal chromosomes into male and female pronuclei, the restoration of the sperm centrosome, and the nucleation of microtubule-mediated motility necessary to bring the male and female pronuclei into close apposition. These events occur after both fertilization in vitro and after intracytoplasmic sperm injection (ICSI), a new technique which is currently being applied in many clinics to overcome severe male infertility. Defects in any of the events leading to fertilization can be lethal to the zygote and may prove to be causes of infertility. METHODS: Imaging of inseminated human and rhesus oocytes using immunohistochemical techniques reveals several phases at which fertilization arrests. RESULTS: Oocytes from some infertile patients failed to complete fertilization due to failure of the sperm aster microtubules in uniting the sperm and egg nuclei. The rate of sperm aster formation, size, and organization during fertilization has been used as a measurement of bovine sperm quality. The development of an assay using Xenopus laevis oocyte extract can also be used to test sperm from various species for their ability to form esters and perform other centrosomal functions in vitro, as well as another indicator of sperm quality. Semen from men with questionable fertility was found to contain sperm which are generally incapable of producing sperm asters. In addition, the activity of centrosomal proteins such as gamma-tubulin and centrin have been detected in mammalian eggs and sperm. The levels of gamma-tubulin increase markedly after exposure to X. laevis egg extract. CONCLUSION: Defects in either male or female nucleus decondensation also resulted in the arrest of fertilization and was found to occur in both inseminated human oocytes and in rhesus oocytes fertilized by ICSI. These discoveries on the molecular basis of infertility in humans have important implications for infertility diagnosis and managing reproduction.

Animals↗
PubMed1996

Phenotypic variations among paternal centrosomes expressed within the zygote as disparate microtubule lengths and sperm aster organization: correlations between centrosome activity and developmental success.

This study describes a paternal effect on sperm aster size and microtubule organization during bovine fertilization. Immunocytochemistry using tubulin antibodies quantitated with confocal microscopy was used to measure the diameter of the sperm aster and assign a score (0-3) based on the degree of radial organization (0, least organized; 3, most organized). Three bulls (A-C) were chosen based on varying fertility (A, lowest fertility; C, highest fertility) as assessed by nonreturn to estrus after artificial insemination and in vitro embryonic development to the blastocyst stage. The results indicate a statistically significant bull-dependent difference in diameter of the sperm aster and in the organization of the sperm astral microtubules. Insemination from bull A resulted in an average sperm aster diameter of 101.4 microm (76.3% of oocyte diameter). This significantly differs (P < or = 0.0001) from the average sperm aster diameters produced after inseminations from bull B (78.2 microm; 60.8%) or bull C (77.9 microm; 57.8%), which themselves displayed no significant differences. The degree of radial organization of the sperm aster was also bull-dependent. Sperm asters organized by bull A-derived sperm had an average quality score of 1.8, which was higher than that of bull B (1.4; P < or = 0.0005) or bull C (1.2; P < or = 0.0001). Results with bulls B and C were also significantly different (P < or = 0.025). These results indicate that the paternally derived portion of the centrosome varies among males and that this variation affects male fertility, the outcome of early development, and, therefore, reproductive success.

Analysis of Variance↗
PubMed1996

Intracytoplasmic sperm injection for Rhesus monkey fertilization results in unusual chromatin, cytoskeletal, and membrane events, but eventually leads to pronuclear development and sperm aster assembly.

The disassembly and reorganization of sperm-derived structures are landmarks for the onset of embryonic development. Since complete information on these events is not yet available, we examined the disassembly of the sperm axoneme, the formation of the sperm aster, and the decondensation and development of the male and female pronuclei in inseminated Rhesus monkey oocytes conceived by in-vitro fertilization (IVF) or by intracytoplasmic sperm injection. During IVF, the spermatozoa lose their acrosomes after contacting the zona pellucida, and the plasma membrane and nuclear envelope disappear after fusion with the oolemma. Subsequently, a sperm aster of microtubules forms around the proximal centriole, which is bound to the sperm connecting piece. This process is then followed by the formation of both pronuclei, which single sperm centriole later duplicates and the bipolar mitotic apparatus is observed. Following sperm injection, the spermatozoa have both an intact plasma membrane and acrosome. Although the microtubules form the sperm aster in a fashion identical to that seen during IVF, the presence of an intact acrosome appears to be associated with a heterogeneity in the decondensation of sperm chromatin. While this may indicate an abnormal pattern of chromatin decondensation during the formation of the male pronucleus following sperm injection, the male pronucleus eventually fully decondenses, as during IVF. Sperm mitochondria are displaced as the sperm centriole is exposed. Annulate lamellae and a previously undescribed organelle which seems to contain annulate lamellae precursors, as well as maternal mitochondria, are found in association with the developing pronuclear envelopes. This information increases understanding of fertilization in primates, and may also be of significance for use in assisted human reproduction as well as in the preservation of endangered mammalian species. In addition, these results demonstrates the similarities between fertilization in Rhesus monkeys and humans, providing additional evidence for the use of this non-human primate as a model system in which to investigate the cellular and molecular biological basis of human reproduction.

Animals↗
PubMed1996

Microtubule and chromatin configurations during rhesus intracytoplasmic sperm injection: successes and failures.

Intracytoplasmic sperm injection (ICSI) was performed on rhesus monkey oocytes, and the resultant microtubule and DNA configurations were imaged by laser-scanning confocal microscopy. In addition, polyspermic oocytes fertilized by ICSI were examined by transmission electron microscopy (TEM). Successful rhesus fertilization by ICSI revealed microtubule and DNA configurations similar to those observed during in vitro fertilization of human and rhesus monkey oocytes, including sperm aster formation, pronuclei decondensation, spindle formation, and cell division. Several abnormalities, however, were also observed: 1) inability to complete meiosis; 2) inability to undergo male or female pronucleus formation; 3) separation of the sperm tail from the sperm nucleus; 4) premature chromosome condensation with the formation of a paternal meiotic spindle; and 5) formation of multiple female pronuclei (karyomeres) during chromosome decondensation. TEM analysis revealed that sperm can undergo decondensation in the presence of an intact acrosome at least 18 h after sperm injection. These results demonstrate the utility of rhesus ICSI in pre-clinical applications as well as with endangered species. However, the different types of fertilization failures observed here indicate that although ICSI may be a readily accepted means of fertilization of human oocytes in many clinics, we should further characterize the cellular and genetic abnormalities associated with ICSI in both human and nonhuman primates.

Animals↗
PubMed1996

Fate of the sperm mitochondria, and the incorporation, conversion, and disassembly of the sperm tail structures during bovine fertilization.

Sperm incorporation and the conversion of the sperm-derived components into zygotic structures during in vitro fertilization of bovine oocytes was explored by combining ultrastructural studies with observations of the fertilizing sperm tagged with a mitochondrion-specific vital dye MitoTracker green FM. The zygotes fertilized by the MitoTracker-labeled sperm were fixed at various times after fertilization and then processed for immunocytochemistry to examine the distribution of DNA, microtubules, and sperm tail components, including the fibrous sheath and axonemal microtubules. We show here that the complete incorporation of the sperm, but not sperm-oocyte binding and oocyte activation, depends upon the integrity of oocyte microfilaments and is inhibited by the microfilament disrupter cytochalasin B. After sperm incorporation, the mitochondria are displaced from the sperm's connecting piece, and the sperm centriole is exposed to the egg cytoplasm. This event is followed by the formation of the microtubule-based sperm aster, which is responsible for the union of male and female pronuclei. Concomitantly, the major structure of the sperm principal piece, the fibrous sheath, disappears. After the first mitosis, the compact mitochondrial sheath can be seen in one of the blastomeres. An aggregate of the sperm mitochondria is observed at the entry of the second mitosis, although they remain in the vicinity of the nucleus and can later be seen at one pole of the metaphase spindle. The mitochondrial cluster is occasionally found in one of the blastomeres in the early-stage four-cell embryos, but it is no longer detected by the beginning of the third mitotic cycle. These data suggest that the disassembly of the sperm tail during bovine fertilization occurs as a series of precisely orchestrated events involving the destruction (fibrous sheath and mitochondrial sheath) and transformation (DNA, sperm centriole) of particular sperm structures into zygotic and embryonic components.

Acrosome↗
PubMed1995

Imaging motility during fertilization.

Studying reproduction in domestic species is now possible at the cellular and molecular level due to advances in the production of large numbers of zygotes and embryos in these species. In this paper we review the microtubule patterns during fertilization in domestic species. These results indicate that domestic species accomplish fertilization in a similar fashion to one another but in a far different fashion from rodents. Recent results indicate that human fertilization is similar to that of domestic species. We discuss the significance this has on the use of domestic species as a model system for human studies and possible consequences for the alleviation of human infertility.

Animals↗
PubMed1995

The sperm centrosome during fertilization in mammals: implications for fertility and reproduction.

This article reviews the recent discoveries that: (1) nearly all mammals, including humans, inherit their centrosomes from their fathers; and (2) some sperm are ineffective in organizing the microtubules essential for effecting genomic union during fertilization, leading to the speculation that these sperm have centrosome defects. In addition, the molecular dissection and reconstitution of the human sperm centrosome in vitro is presented.

Animals↗
PubMed1994

Microtubule organization in the cow during fertilization, polyspermy, parthenogenesis, and nuclear transfer: the role of the sperm aster.

Microtubule organization in bovine oocytes during fertilization, polyspermy, parthenogenesis, and nuclear transfer is examined with the goal of understanding microtubule activity and the manner in which the centrosome during fertilization and mitotic spindle poles are established. In the unfertilized bovine oocyte, microtubules are detected only in the metaphase-arrested second meiotic spindle; no cytoplasmic asters are observed. After insemination, a small aster of microtubules is seen adjacent to the incorporated sperm head. This aster enlarges and, at the time of pronuclear apposition, fills the cytoplasm. At prophase, the aster splits and forms the poles for the first mitotic spindle, which is anastral, fusiform, and often located eccentrically. During anaphase, asters assemble at each spindle pole. After telophase, these asters develop into the interphase array of microtubules in the daughter blastomeres. During polyspermy, an aster forms from a site between each incorporated sperm head and tail. Multiple mitotic spindles are observed in polyspermic zygotes; multipolar spindles are not seen. Parthenogenetic activation with 5 microns ionomycin followed by a 4-hr incubation in 1.9 mM dimethylaminopurine results in > 80% activation, and antitubulin immunofluorescence microscopy demonstrates that initially disarrayed microtubules are observed and the some microtubules extend from the remnants of the second meiotic spindle. At the time normal for cell division, these parthenogenotes form anastral, barrel-shaped bipolar mitotic spindles. Asters form at the spindle poles at anaphase and the parthenogenotes divide from one to two blastomeres. Nuclear fusion of a morula-derived blastomere to a parthenogenetically activated oocyte results in either a single microtubule aster or at times, two asters, formed in association with the donated nucleus. These results demonstrate that fertilization in the cow is a blending of paternally and maternally derived centrosomal material.

Animals↗
PubMed1994

Microtubule configurations in oocytes, zygotes, and early embryos of a marsupial, Monodelphis domestica.

The marsupials represent a separate evolutionary lineage from eutherians from which they diverged over 100 million years ago. In order to explore the origin and mode of centrosome inheritance amongst this group of mammals, this study investigates the microtubule organization during fertilization, parthenogenesis, and polyspermy in the didelphid, Monodelphis domestica. Microtubules and DNA were visualized in maturing ovarian oocytes, parthenogenetically activated oocytes, monospermic and polyspermic zygotes, and early embryos. Ovarian oocytes had a central region of yolky cytoplasm that, after fertilization, became polarized; much of the yolk was then extruded into the perivitelline space as an enucleated cytoplasmic mass. Immunofluorescence microscopy, using a monoclonal antibody to beta-tubulin, demonstrated microtubules in the meiotic spindle in unfertilized oocytes, but cytasters were not detected. After fertilization, a cluster of microtubules forming into a sperm aster was evident around the male pronucleus. The sperm aster remained largely restricted to the nonyolky region of the egg cytoplasm, resulting in a cytoplasmic heterogeneity between a microtubule-rich region and one in which microtubules were largely absent. Once the two pronuclei came close together, abundant microtubules were found surrounding both pronuclei. In the early embryo, microtubules were found in the outer cortical region of the blastomeres and, in addition, there was an extensive and elaborate network of microtubules throughout the yolk mass. Disruption of the meiotic spindle microtubules with nocodazole or cold treatment did not result in chromosome dispersion in the cortex and recovery from drug or cold depolymerization demonstrated that microtubules might not be as dynamic as those in eutherian mammals. Taxol stabilization resulted in an increase in cortical microtubules. In this marsupial species, therefore, the centrosome appears to be of paternal origin, and the radiating microtubules that form may well be involved both in bringing the pronuclei together and in the cytoplasmic polarization that results in extrusion of the yolk mass.

Animals↗