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PubMed2001

The nuclear location and chromatin organization of active chorion amplification origins.

It remains unclear how certain regions on metazoan chromosomes are selected to initiate DNA replication. In recent years a number of origins of DNA replication have been mapped, but there is still no DNA consensus for predicting where replication will initiate. Evidence suggests that the higher order structure of the nucleus and chromosome influences origin activity. Chromosomal DNA replication is proposed to occur in special compartments in the nucleus called replication foci. Foci in different regions of the nucleus initiate replication at different times of S-phase, suggesting nuclear position may contribute to where and when replication begins. Here we test the contribution of nuclear compartments for well-defined origins, those involved in amplification of the chorion (eggshell) genes during Drosophila oogenesis. The results of three-dimensional confocal microscopy indicate that chorion DNA replication origins are highly active in diverse positions within the nucleus. We also find that chorion replication origins inserted at ectopic chromosomal sites can amplify highly in diverse nuclear locations distinct from the endogenous loci, including when they are buffered against genomic position effects. We used fluorescence in situ hybridization to analyze chromosome structure during amplification. Contrary to the replication factory model, we find no evidence for spooling of DNA toward a replication center. We discuss the implications of these results for understanding the role of higher order structure in amplification and chromosome duplication.

Animals↗
PubMed2003

Detection of P-glycoprotein-mediated multidrug resistance against anthelmintics in Haemonchus contortus using anti-human mdr1 monoclonal antibodies.

The "multidrug resistance" (MDR) system involves the action of transmembrane P-glycoproteins (Pgp) which may be responsible for failure of chemotherapy in both invertebrates and vertebrates. We previously obtained partial reversion of anthelmintic resistance in nematodes subjected to both anthelmintics and inhibitors of this system. The results presented here are able to describe more accurately the presence of Pgp in nematodes because of the use of C219 and UIC2 monoclonal antibodies, which are used for the detection of human and mouse mdr1 gene products. These antibodies demonstrated the presence of Pgp in eggshells. Their role in these structures, which are considered to be passive barriers, remains to be determined. Flow cytometry analyses of the UIC2 staining allowed determination of the resistance of individuals, which varied within the parasite population. UIC2 demonstrated both the presence and activity of Pgp in nematodes as has previously been shown in tumour cells. Resistance seems to be due to an increase in both the number of Pgp sites and parasites with high levels of Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Experimental schistosomiasis, protective aspects of granulomatous reaction in the mouse liver.

We show two mechanisms of liver protection by the granulomatous reaction against Schistosoma mansoni eggs entrapped in the organ. First, eosinophil peroxidase and its substrate H(2)O(2) are released by inflammatory cells in the immediate vicinity of the parasite eggs. The efficiency of this process was demonstrated by administration of antioxidants to infected mice. The treatment, which reduces H(2)O(2) production, significantly improved the ability of parasite eggs to hatch after collection from the liver. Secondly, we labeled the released egg antigens in liver histological sections and we found that the lattice of collagen fibers which is built around eggs appears to create a barrier preventing released compounds from diffusing freely in surrounding tissues. Together, oxidative processes and antigen containment allow the parasitized liver to cope with the dual threat posed by parasite eggs, i.e. a highly resistant chitinous eggshell and the release of toxic substances.

Animals↗
PubMed2002

Dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis.

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.

Animals↗
PubMed2005

Annexins as cell-type-specific markers in the developing chicken chorionallantoic membrane.

Between day E8 and E12 of embryonic development, the chicken chorioallantoic membrane (CAM) undergoes massive structural rearrangement enabling calcium-uptake from the eggshell to supply the growing embryo. However, the contribution of the various cell types of the chorionic epithelium including the capillary covering (CC) cells, villus cavity (VC) cells, endothelial-like cells, and basal cells to this developmental program is largely unknown. In order to obtain markers for the different cell types in the chorionic epithelium, we determined the expression patterns of various calcium-binding annexins in the developing chicken CAM. By reverse transcription/polymerase chain reaction with primers deduced from nucleotide sequences available in various databases, the presence of annexin (anx)-1, anx-2, anx-5, and anx-6 was demonstrated at days E8 and E12. Quantitative immunoblotting with novel antibodies raised against the recombinant proteins revealed that anx-1 and anx-5 were significantly up-regulated at day E12, whereas anx-2 and anx-6 expression remained almost unchanged in comparison to levels at day E8. Immunohistochemistry of paraffin-embedded sections of E12 CAM revealed anx-1 in CC cells and VC cells. Anx-2 was localized in capillaries in the chorionic epithelium and in basal cells of the allantoic epithelium, whereas anx-6 was detected in basal cells or endothelial-like cells of the chorionic epithelium and in the media of larger vessels in the mesenchyme. A 2-day exposure of the CAM to a tumor cell spheroid resulted in strong proliferation of anx-1-expressing CC cells suggesting that these cells participate in the embryonic response to experimental intervention. Thus, annexins exhibit complementary expression patterns and represent appropriate cell markers for the further characterization of CAM development and the interpretation of results obtained when using CAM as an experimental model.

Animals↗
PubMed2006

High jugular bulb in the translabyrinthine approach to the cerebellopontine angle: anatomical considerations and surgical management.

BACKGROUND: Evidence of a high jugular bulb position (HJBP) during the translabyrinthine approach may compromise the surgical removal of cerebellopontine angle (CPA) tumours. We report a simple surgical procedure to safely manage this frequent normal variation and comment on various alternative options. METHODS: The translabyrinthine approach included a complete skeletonization of the sigmoid sinus and of the presigmoid dura. A thin eggshell bone was left at the jugular bulb surface. The dome of the jugular bulb was gently dissected from the jugular fossa and gradually retracted downward in a tailored way, allowing the surgeon to drill below the internal auditory meatus. A small piece of bone was wedged over the jugular dome in order to maintain its lowered position. RESULTS: Among 178 consecutive translabyrinthine approaches performed for the removal of large CPA tumors, the use of this procedure was required in 44 cases of HJBP. Excepting minimal venous bleeding easily controlled in several cases, we never observed any complication from this procedure nor failure to expose the inferior compartment of the CPA. CONCLUSIONS: The HJBP can be systematically diagnosed with the preoperative CT-scan using bone window imaging. Our results demonstrate that the described procedure is safe and effective to widen the operative corridor that is required for the exposure of the inferior compartment of the CPA in this anatomical situation.

Adult↗
PubMed1999

Ossifying pituitary gonadotroph adenoma: A case report.

BACKGROUND: Bone formation in pituitary adenomas is a rare finding. Only two previous cases were published, occurring in a prolactin- producing and a growth hormone- producing pituitary adenoma respectively, both in pre-menopausal women. CLINICAL MATERIAL: This is the third report of an ossified pituitary adenoma and the first report of a pituitary gonadotroph adenoma with bone formation occurring in an elderly man. MRI imaging revealed an unusual eggshell cap-like calcified structure surrounding the tumor. Histologically, the adenoma contained irregularly anastomosing trabecules with well formed lacunae and osteoblasts along the margins. CONCLUSIONS: Insufficient tumor blood supply may trigger proliferation of connective tissue that subsequently undergoes osteoid metaplasia. Pituitary adenoma with osteoid metaplasia should be included in the differential diagnosis of calcifying tumors in the sella region.

Adenoma↗
PubMed2005

Metal concentrations, foraging distances, and fledging success of great blue herons nesting along the Hanford Reach of the Columbia River.

An ecological risk assessment of the spatial distribution of metal concentrations along the Hanford Reach of the free-flowing Columbia River in southcentral Washington, identified great blue herons, Ardea herodias, at potential risk through the ingestion of contaminated riverine biota, especially fish. We measured metal concentrations in livers of pre-flight herons from the Hanford Reach and excrement samples taken from the same nests. Nests were distributed among three colonies situated upstream and downstream from nine retired plutonium production reactors along the river where metals in reactor coolant waters had been released directly into the river or disposed to shoreline retention basins and ditches. Distances traveled by parent herons to foraging areas along the river shore were determined by visually tracking parent birds as they flew from nests to upriver and downriver foraging sites. Foraging flight distances varied between colonies with mean distances ranging between 0.7 and 3.1 km. Cadmium, Cr, and Pb concentrations were higher in excrement than in the livers of pre-flight herons but the opposite was noted for Cu, Hg, and Zn. Highest metal concentrations of Cr, Cu, Zn, Cd, and Pb, were measured in excrement taken from heron nests at the colony located upstream from all reactors. These results were consistent with metal concentrations reported in river sediment from the same regions, indicating excrement from the heron nests may be a useful indicator of dietary uptake of metals by herons. Fledging success and eggshell thickness measurements were used as an index of health of the local heron population. The results indicate that the reproductive health of great blue herons nesting along the Hanford Reach is among the highest reported in the continental United States.

Animals↗
PubMed1983

Effect of experimentally induced calcium deficiency on the developmental expression of collagen types in chick embryonic skeleton.

In order to investigate the effect of embryonic calcium deficiency on the cellular differentiation processes in embryonic skeletogenesis, chick embryos were maintained in long-term shell-less cultures in vitro. The absence of the eggshell, which normally provides over 120 mg of calcium to the embryo during the course of development, resulted in severely retarded and anomalous skeletal formation. The pattern of cytodifferentiation in the skeletal elements during development was assessed by examining collagen type synthesis in both endochondral and intramembranous bones of normal and shell-less embryos as a function of developmental age. Skeletal tissues obtained from these embryos at various developmental stages were maintained in short-term organ culture in medium containing [3H]Pro. The metabolically labeled collagen was isolated from these tissues and typed biochemically based on electrophoresis, ion-exchange chromatography, differential salt fractionation, zone precipitation chromatography, and CNBr peptide mapping. The results indicate that, compared to chronologically equivalent normal controls, calcium-deficient skeletal elements from shell-less embryos appeared to fail to mature into complete bony tissues and instead exhibited partial cartilage phenotype with the expression of cartilage-specific type II collagen.

Age Factors↗
PubMed1985

Control of cell-cycle timing in early embryos of Caenorhabditis elegans.

A technique has been developed for extruding either substantial amounts of cytoplasm without nuclei or individual nuclei with small amounts of cytoplasm from early embryos of C. elegans after perforating the eggshell with a laser microbeam. This technique, in conjunction with laser-induced cell fusion, has allowed the altering of nuclear/cytoplasmic ratios and the exposing of the nucleus of one cell to cytoplasm from another. Using these approaches the roles of nuclei and cytoplasm in determining the different cell-cycle periods of the several blastomere lineages in early embryos have been examined. It was found that nuclei in a common cytoplasm divide synchronously; enucleated blastomeres retain a cycling period characteristic of their lineage; cycling period is not substantially affected by changes in the ratio of nuclear to cytoplasmic volumes or the DNA content per cell; the period of a cell from one lineage can be substantially altered by introduction of cytoplasm from a cell of another lineage with a different period; and short-term effects of foreign cytoplasm on the timing of the subsequent mitosis differ depending on position of the donor cell in the cell cycle. These results are discussed in connection with models for the action of cytoplasmic factors in controlling cell-cycle timing.

Animals↗
PubMed1988

Organization and expression of a second chromosome follicle cell gene cluster in Drosophila.

Four genes expressed during the period of vitelline membrane formation are clustered within 8 kb of DNA in region 26A of the second chromosome. Temporal and quantitative difference in the profiles of accumulated RNA suggest that the genes are independently regulated although they are selectively expressed during the stages of vitelline membrane biosynthesis. In situ hybridization and S1 analyses of RNAs from fractionated eggchambers established that these genes are active only in the follicle cells. S1 mapping with in vitro synthesized RNA probes shows that three of the genes are tandemly oriented. All four appear to be intronless. In vitro translation products from hybrid-selected RNAs indicate that two of these genes code for major vitelline membrane proteins. Sequence analysis of these two genes support this conclusion. The cell- and stage-specific expression of the other two genes, encoding less abundant RNAs, suggests that they also play a role in early eggshell production.

Amino Acid Sequence↗
PubMed1989

A sperm-supplied product essential for initiation of normal embryogenesis in Caenorhabditis elegans is encoded by the paternal-effect embryonic-lethal gene, spe-11.

Loss-of-function mutations in the spe-11 gene in Caenorhabditis elegans result in a paternal-effect embryonic-lethal phenotype: fertilization of wild-type oocytes by sperm from homozygous spe-11 mutant males leads to abnormal zygotic development, whereas oocytes from homozygous spe-11 hermaphrodites when fertilized by wild-type sperm develop normally. Embryos fertilized by sperm from homozygous spe-11 worms fail to complete meiosis and show defects in eggshell formation, mitotic spindle orientation, and cytokinesis. Genetic analysis suggests that the spe-11 gene is expressed before the completion of spermatogenesis and that the wild-type locus encodes a product that is present in sperm and participates, directly or indirectly, in initiating the correct program of early events in C. elegans embryos. Such an ontogenetic role of the spe-11+ gene product in early embryogenesis distinguishes spe-11 mutations from the two paternal-effect mutations identified in Drosophila, ms(3)K81 and pal, which primarily affect chromosome behavior. Analysis of spe-11 provides the first step toward genetic dissection of the functions of the sperm in early embryogenesis in C. elegans.

Animals↗
PubMed1991

Expression during Drosophila development of DER, a gene related to erbB-1 and neu: correlations with mutant phenotypes.

We used in situ hybridization to study expression of the DER gene during Drosophila development. DER encodes a transmembrane cell-surface receptor with a cytoplasmic protein-tyrosine kinase domain, and resembles the vertebrate genes that encode the EGF receptor and the neu protein. We examined most stages of development in the Drosophila life cycle and found a substantial correlation between DER expression and the phenotypes associated with a variety of mutant alleles. Of particular note were high levels of expression in the primordia of the mouth parts, which are the embryonic tissues most sensitive to reductions in DER activity; discrete expression in a subset of neural cells essential for construction of the axonal scaffold, a structure that is deformed in mutant embryos; uneven expression in the eye disc, the development of which is abnormal in both mild hypomorphs and hypermorphs; and expression in the follicular epithelial cells of the ovary, which are responsible for producing the eggshell of developing oocytes and do so aberrantly in the mildest hypomorphs. However, DER transcripts were also detected in a subset of tissues that have not been reported to be abnormal in mutant organisms. Our findings agree with and extend recently reported results for the DER protein, indicating that DER is regulated primarily at the level of transcription, in contrast to previous suggestions. We conclude that the phenotypes displayed by recessive mutants can be attributed to loss of DER function in the affected tissues.

Animals↗
PubMed1992

Mutant yolk proteins lead to female sterility in Drosophila.

Specific mutations in the yolk protein genes, yp1 and yp2, of Drosophila melanogaster cause the yolk proteins (YPs) they encode to precipitate, ultimately resulting in female sterility. YPs of the yp1 mutant fs(1)1163 are secreted normally but then precipitate as globules and occasionally as crystalline fibers in the subbasement membrane space of the fat body (Butterworth et al., 1991, J. Cell Biol. 112, 727-737). The present ultrastructural and immunological studies of the fat body of the yp2 mutant fs(1)K313 show that YP also precipitates as globules in the same tissue compartment. The globules are also incapable of passing into the hemolymph but they are morphologically distinct from those of fs(1)1163. Similar analyses were performed on developing oocytes in wild type and both mutant strains. YP-containing aggregates, ultrastructurally similar to those in the fat body of each respective mutant, were found in the space between the plasmalemma and the vitelline membrane and embedded within the membrane itself. The evidence suggests that the precipitates interfere with the correct assembly of the eggshell membranes, leading to the sterile phenotype. Immunogold studies demonstrate that newly synthesized YPs in the normal and mutant strains share secretory vesicles with putative, vitelline membrane proteins and that the translocation of follicle cell YP is not through the membrane along the interfollicular spaces but directly through the plasmalemma facing the oocyte. Further the YP precipitates in the mutants permit visualization of the polarity of exocytosis of YP from the follicle cells.

Animals↗
PubMed1987

Relationship of early neural tube appearance to myeloschisis in a chick embryo model.

Myeloschisis, a form of neural tube defect involving the spinal cord, was induced in chicks by creating a window in the eggshell of the fertilized egg at 26 h after placement in an incubator. The embryos were stained and photographed through the window at 48 to 60 h after commencement of incubation and the neural tube was determined to be normal, delayed, irregular, or abnormally open for the developmental stage of the embryo. The eggs were then reincubated until 14 days of age. At that time the chicks were determined to have normal spinal cords or myeloschisis by gross examination, dissection, and histology. The appearance of the neural tube at 48 to 60 h was then correlated with the appearance of the spinal cord at 14 days of age. It was determined that in the chick embryo model of myeloschisis induced by windowing, a normal neural tube at the time of neural tube closure results in a 2.3% incidence of myeloschisis, a widely open neural tube a 47% incidence of myeloschisis, an irregular neural tube a 37% incidence of myeloschisis, and delayed closure of the neural tube a 14% incidence of myeloschisis. Thus, no appearance of an early neural tube can be said to always correlate with myeloschisis or a normal outcome. Additionally, it appears that within a single model there may be multiple mechanisms of production of myeloschisis.

Animals↗
PubMed1985

Trichostrongylus colubriformis: egg lethality due to Bacillus thuringiensis crystal toxin.

A toxin from crystals of Bacillus thuringiensis israelensis was lethal in vitro to eggs of the ruminant nematode Trichostrongylus colubriformis, with an LD50 of 1.8 ng/ml. Larval viability declined after a 2-hr exposure to B. t. israelensis and was dependent on the development period of eggs prior to exposure. Alkaline solubilization suggested that the insecticidal delta-endotoxin of B. t. israelensis was not responsible for nematicidal activity. Filtration of the toxin through 0.2- or 0.45-micron-pore filters revealed that the nematicidal activity was retained on the filter. Toxicity for nematode eggs was decreased by the enzyme inhibitor L-1-tosylamide 2-phenylethylchloromethyl ketone (10(-4) M) or ethylenediaminetetraacetic acid (10(-5) M) and phenylmethylsulfonyl fluoride (10(-6) M). Ethylenediaminetetraacetic acid from 10(-9) to 10(-5) M had no effect on the toxicity while phenylmethylsulfonyl fluoride from 10(-9) to 10(-5) M inhibited toxicity. Fourteen mammalian and microbial enzymes had no significant effect on larval viability while 12 sugars and lipids failed to reduce the toxicity. Addition of 5 mM calcium to the eggs' medium decreased the B. t. israelensis toxicity by 20-fold. The calcium-dependent inhibition of toxicity was reversed by ethylenediaminetetraacetic acid (10(-5) M) and lanthanum chloride (100 microM). The ionophore A-23187 decreased the LD50 by 18-fold to 33.5 ng/ml. Addition of 5 mM calcium chloride to the ionophore and toxin yielded an LD50 of 9.2 ng/ml. Treatment of nematode eggs with B. t. israelensis toxin for 2 or 24 hr had no effect on subsequent binding of selected fluoresceinated lectins to the eggshell.

Animals↗
PubMed1986

The effects of a calcium deficient diet on the mechanical properties and morphology of goose bone.

A control group of geese (Anser anser) on a normal calcium diet for egg laying poultry was compared to egg laying geese on a calcium deficient diet. The ultimate compressive strength and modulus of elasticity of femoral cortical bone from each group were determined by compressing right circular cylinders which were 2.4 mm in height and 0.8 mm in diameter. The bending strength and bending modulus of elasticity of tibial cortical bone were determined by three point bend tests on rectangular prisms which were approximately 25 mm by 0.8 mm by 0.8 mm. Bone calcium content and eggshell calcium content were determined by atomic absorption spectrophotometry. Blood samples were analyzed for free calcium ion concentration. Histological observations included studies of cross-sectional microradiographs, examinations of cross sections stained by a modified Masson's technique, and a determination of fractional area of voids by quantitative microscopy. The average compressive modulus for the control birds was 12.0 GPa (S.D.: 6.2 GPa) while the ultimate compressive strength was 165 MPa (S.D.: 27 MPa). Calcium deprived birds showed slight, but not statistically significant, decreases in both the compressive modulus and compressive strength. The tibial three point bending modulus for the control birds was 16.5 GPa (S.D.: 2.6 GPa) while the ultimate bending strength was 256 MPa (S.D.: 58 MPa). Once again, slight though not statistically significant decreases in the bending modulus and strength were seen in the geese on the calcium deficient diet. The average calcium content (wt%) of the femora of the control birds was 20.5% (S.D.: 4.3%) and 20.6% (S.D.: 4.8%) for the tibiae. No significant differences were noted in the calcium deprived birds. The average fractional void area for the control bird femoral bone was 12.0% (S.D.: 2.6%) and 9.8% (S.D.: 1.8%) for the tibial bone. Significantly greater fractional void areas were noted in the calcium deficient birds as were profound changes in the macrocellular structure of these bones.

Animals↗
PubMed1976

Effects of ambient pressures, He and Sf6 on O2 and CO2 transport in the avian egg.

The CO2 and O2 tnesions were determined in the air cells of 14-16 day old chicken eggs before and after transfer to a 21% O2 in He or SF6 atmosphere. In the former gas mixture the air-cell PCO2 (which reflects the arterialized blood PCO2) fell rapidly from 32 torr in air to 17 torr in He-O2 attaining a new steady state in 2-4 h. In the SF6-O2 mixture PCO2 rose from 36 torr in a similar period. A similar PCO2 increase was also observed when eggs were compressed to 2 atm of air while exposure to 0.5 atm with a 40% O2-N2 mixture decreased PCO2. Since gas transport across the eggshell is by gas phase diffusion, these findings can be explained by the changes in the diffusion coefficient of CO2 which increase in the presence of He and decrease in SF6. Furthermore, the diffusion coefficient is also inversely related to the absolute pressure. Quantitative prediction of the changes of PCO2 in the He and SF6 mixture cannot be made, since the binary diffusion coefficients are not necessarily applicable in a ternary gas mixture. However, effective diffusion coefficients in these multicomponent mixtures can be derived on the basis of the observed PCO2 changes.

Animals↗