bluuebunny/crossref_metadata_2025 huggingface dataset:
Release of metaphase arrest by partial inhibition of protein synthesis in blue mussel oocytes
Authors: Dubé Francois , Dufresne Louise
Published: June 2005

In the blue mussel Mytilus edulis, shed oocytes are arrested at metaphase I of meiosis until fertilization. In this study, the mechanisms involved for maintaining the oocytes in metaphase were investigated. Analysis of 35‐methionine‐labelled proteins separated by 1D SDS‐PAGE reveals that two protein bands of apparent MW of 50 and 54 kDa periodically appear and disappear during meiotic and mitotic cycles. Moreover, the 50 kDa protein band, clearly detected in unfertilized oocytes, is seen to disappear within 15 min after fertilization. Unfertilized or fertilized oocytes treated with emetine have a protein synthesis rate reduced to less than half the normal rate observed in the untreated cells. Addition of this protein synthesis inhibitor at various times after fertilization inhibits the normal progression through the cell cycles. However, emetine added to unfertilized oocytes induces the completion of first meiotic maturation, polar body extrusion, and the decondensation of chromosomes which form one or two large pronuclei. This process is accompanied by DNA synthesis, and is preceded by the early disappearance of the 50 kDa protein band, seen to cycle after fertilization. These results indicate that metaphase arrest, in mussel oocytes, requires the continuous synthesis of short‐lived proteins, the destruction of which is sufficient to induce meiosis resumption followed by DNA synthesis.
Sex‐determining potencies vary among female incubation temperatures in a turtle
Authors: Bull J. J. , Wibbels T. , Crews D.
Published: June 2005

Reptiles whose sex is determined by incubation temperature typically exhibit all‐male or all‐female sex ratios over a wide range of incubation temperatures. The question arises as to whether the various all‐female temperatures (or the various all‐male temperatures) are equivalent in their “potency,” or capacity to effect female determination. In map turtles, warm incubation temperatures produce all females and cool ones produce all males. We compared sex determining potencies of two all‐female temperatures, 31°C and 32.5°C, by incubating eggs first at a male‐producing temperature (26°C) and then shifting them to the warm temperatures. The resulting sex ratio was significantly more male biased in the 26°C→31°C shift than in the 26°C→32.5°C shift, indicating that 32.5°C has the greater female potency. These results point to the possibility that sex determination depends on a quantitative rather than qualitative level of gene expression.
Expression of the Axd (axial defects) mutation in the mouse is insensitive to retinoic acid at low dose
Authors: Haviland Martha B. , Essien Francine B.
Published: June 2005

The Axd mutation in the mouse acts by an unknown mechanism to cause lum‐bosacral open neural tube defects and a variety of tail anomalies. Retinoic acid (RA) plays a number of different physiological and developmental roles and has been shown to affect neurulation in mice and other species. Indeed, reports have shown that this biologically active compound (or its metabolites) at low dose can alter the incidence of neural tube defects (NTD) in curly‐tail (ct), splotch (Sp), and delayed splotch (Spd) mice, strains that are genetically predisposed to such abnormalities. The aim of the present study was to determine if RA administered under similar conditions would affect the penetrance or expression of the Axd mutation or survival of Axd homozygotes. Axd/+ and +/+ dams were exposed to RA intraperitoneally (5 mg/kg) on D9 postcoitus. No difference in incidence or extent of neural tube defects or other axial anomalies was detected among embryos of Axd/+ dams given RA compared with those administered vehicle only. This finding is consistent with the diversity of gene‐controlled steps required for neurulation and the differing sensitivities of specific mutants to rescue by extrinsic agents.
Stimulation of 35S‐sulfate uptake by mammalian insulin‐like growth factors I and II in cultured cartilages of the Japanese eel, Anguilla japonica
Authors: Duan Cunming , Hirano Tetsuya
Published: June 2005

Effects of mammalian insulin‐like growth factors I and II (IGF‐I and II) on 35S‐sulfate uptake by the branchial cartilage of the eel were examined in vitro. Addition of bovine IGF‐I to the medium stimulated sulfate uptake by the cartilage from hypophysectomized eels in a dose‐dependent manner (10–1,000 ng/ml). The cartilages from intact eels responded to IGF‐I similarly. Rat IGF‐II was about 50 times less potent than bovine IGF‐I in stimulating the sulfate uptake by cartilages from intact eels. These results show that sulfate uptake by eel cartilage is stimulated by a mammalian IGF‐I‐like principle and raise the possibility of regulation by a similar principle in teleosts.
Enhancement of aster‐induced furrowing activity by a factor associated with the nucleus
Authors: Rappaport R.
Published: June 2005

The purpose of this investigation was to determine whether asters that develop in the absence of nuclear material have the normal ability to cause surface contraction and furrows. Experimental cells were confined in silicone rubber tubes with flattened lumens. The lumen roof was thin enough to permit deformation or perforation of the contained eggs by pressure with glass tools. When a flattened, elongate egg was perforated at its midpoint with a relatively large glass ball before nuclear membrane breakdown, the positions of the asters and nucleus were determined by chance. When the nucleus and asters were on the same side of the perforation, normal furrows developed. When the nucleus was separated from the aster pair, only ephemeral furrows developed. When the asters straddled the perforation so that only one of the asters was associated with the nucleus, ephemeral furrowing activity was most pronounced at the nucleated end of the cell.

The asters of cells that were kneaded before insertion in the flattened capillary were often farther apart than normal, and the nucleus was more closely associated with one aster than the other. Constrictions developed in the planes of both asters, but only those associated with the aster closest to the nucleus persisted to produce two aster‐containing cells, only one of which contained a nucleus. In subsequent division cycles, asters reproduced and developed in both cells, but only the progeny of the nucleated cell formed permanent furrows. The normal furrow‐inducing capacity of the asters appears to depend in part on the presence of a factor associated with the nucleus.
The expression of nuclear lamin A and C epitopes is regulated by the developmental stage of the cytoplasm in mouse oocytes or embryos
Authors: Prather R. S. , Kubiak J. , Maul G. G. , First N. L. , Schatten G.
Published: June 2005

The cytoplasmic regulation of changes of nuclear lamin antigens was examined by transferring 16‐cell stage blastomeres into mouse oocytes. Sixteen‐cell stage blastomeres were transferred to either pronuclear eggs, enucleated pronuclear eggs or metaphase II oocytes, which were subsequently activated. Pronuclei react with a monoclonal antibody to A/C lamins (J9), whereas nuclei from 16‐cell stage blastomeres do not react with J9. However, after transfer of 16‐cell stage nuclei to activated metaphase II oocytes, the transferred nuclei acquire the antigen. This is in contrast to 16‐cell nuclei that were transferred to intact or enucleated pronuclear eggs; i.e., the nuclei only faintly acquired the A/C epitope. These results suggest that the developmental stage of the cytoplasm regulates the exposure of nuclear lamina epitopes, perhaps by limiting the supply of lamin A/C in the oocyte or because nuclear lamina assembly can only occur at the telophase transition. Furthermore, it appears that there is some exchange of the A/C epitope between (pro)nuclei within the same cell but that the majority of the A/C lamin epitope can be removed from a cell with (pro)nuclear removal.
Drosophila alcohol dehydrogenase: A novel reporter gene for use in mammalian embryos
Authors: Nielsen Loretta L. , Pedersen Roger A.
Published: June 2005

A reporter gene construct containing the Drosophila alcohol dehydrogenase (ADH) gene under regulation of the Rous sarcoma virus long terminal repeat (RSV LTR) was microinjected into mouse zygote pronuclei. ADH activity after injection of the RSV‐ADH construct was visualized in cultured embryos by means of a simple histochemical enzyme assay. The RSV LTR was an efficient promoter that led to abundant ADH activity at all stages of preimplantation development. There was a high incidence of mosaicism among stained embryos.
Haemolymph protein as a source for free amino acid accumulation in the pulmonate Helisoma trivolvis during hypertonic stress
Authors: Matsushima Osamu , Khan Hamid R. , Saleuddin A. S. M.
Published: June 2005

The metabolic origin of hypertonically induced intracellular free amino acid (FAA) accumulation in the freshwater pulmonate snail, Helisoma trivolvis was examined. The changes in FAA patterns in response to hypertonic stress were common in haemolymph and tissues; but FAA concentrations were much lower in the former. Transport experiments with 3H‐glycine and detection by high‐performance liquid chromatography (HPLC) showed that isolated kidneys in vitro took up FAA against a concentration gradient. Fluorography demonstrated that injected 14C‐amino acids were incorporated into haemolymph protein significantly less in hypertonically stressed snails than in control snails. These results suggest that, in H. trivolvis, reduction in synthesis of haemolymph protein and FAA transport from haemolymph into cells contribute to intracellular FAA increase during the initial stage of hypertonic stress.
Energy consumption in the perfused gills of the euryhaline crab Eriocheir sinensis [H. Miln. Edw. ] adapted to freshwater
Authors: Welcomme L. , Devos P.
Published: June 2005

The euryhaline crab Eriocheir sinensis adapted to freshwater shows an ion transporting‐type epithelium in the three posterior gills and a respiratory‐type epithelium in the three anterior gills. When they are perfused in freshwater conditions, there is an increased transport of ions principally in the posterior gills.

We also measure an increased degradation of the branchial glycogen. This polysaccharide is present in an extraepithelial lamina located in the hemal space of the lamellae. Its localization outside the ion‐transporting epithelium and its higher consumption by the posterior gills submitted to hypo‐osmotic conditions suggest a well‐suited glucose transport mechanism through their epithelial membrane. Accordingly, the uptake of glucose by the epithelial cells is slightly higher in the posterior gills as compared with the anterior.
Response of hepatic thyroxine 5′‐deiodinase of rainbow trout, Oncorhynchus mykiss, to chronic ingestion of 3,5,3′‐triiodo‐L‐thyronine
Authors: Eales J. G. , Finnson K. R.
Published: June 2005

Rainbow trout held at 6.5°C were fed food supplemented with 12 ppm T3 (3,5,3′‐triiodo‐L‐thyronine). By 2 weeks, plasma T3 had increased from 3.7 ng/ml (controls) to 12 ng/ml and stayed at that level for 12 weeks with minor change in plasma L‐thyroxine (T4). The hepatic microsomal 5′‐deiodinase (5′‐D) activity (Vmax fell to 25% of control values by 2 weeks and by 12 weeks was undetectable. Trout were also fed the T3‐supplemented food for 6 weeks and then reverted to control food (0 ppm T3) for 6 weeks. After 2 weeks reversion, plasma T3 was 48% lower and 5′‐D Vmax 78% lower than for control fish fed 0 ppm T3 throughout; after 6 weeks reversion, plasma T3 had recovered, but 5′‐D had regained only 58% of control values. These data confirm that exogenous T3 suppresses endogenous T3 production and suggest extrathyroidal autoregulation of T3 production. Furthermore, even 6 weeks after T3 treatment had ceased, hepatic 5′‐D was subnormal, indicating development of T3 dependency.
Phosphoenolpyruvate carboxykinase from ribbed mussel gill tissue: Reactivity with metal ions, kinetics, and action of 3‐mercaptopicolinic acid
Authors: Harlocker Susan L. , Kapper Martin A. , Greenwalt Dale E. , Bishop Stephen H.
Published: June 2005

Phosphoenolpyruvate carboxykinase (E.C. 4.1.32) from ribbed mussel gill cytosol exists as a monomer with Mr 70,000. Low levels of Mn2+ or Zn2+ were required for activity when assayed at high physiological Mg2+ levels. Mg2+ alone would not activate the enzyme. Optimal activity was observed at pH 6.6. The apparent Kms for phosphoenolpyruvate (PEP) and NaHCO3 were 396 μM and 13 mM, respectively, when assayed at pH 6.8 in the presence of 1 mM Mn2+, 1 mM Mg2+, and 1.5 mM IDP. GDP (Kmapp 24 μM) and IDP (Kmapp 46 μM) were both reactive; other nucleoside diphosphates were not. Under standard assay conditions at pH 6.8, the reaction was freely reversible with the rate of PEP carboxylation two to three times the rate of OAA decarboxylation. When assayed in the PEP synthesizing direction at pH 6.8 with 2 mM Mn2+ and Mg2+, the apparent Kms for GTP and OAA were 55 μM and 24 μM, respectively. Chloride ion was inhibitory (I50 120 mM); 3‐mercaptopicolinic acid (MPA) and 3‐aminopicolinic acid (APA) were competitive inhibitors with respect to PEP and noncompetitive with respect to IDP. MPA was a more effective inhibitor (Kiapp 8–34 μM) than APA (Kiapp 250–500 μM). Aminooxyacetate and N‐butylmalonate (BMA) were not inhibitory and hydroxymalonate was slightly inhibitory (Kiapp 15 mM). ATP showed mixed competitive inhibition with respect to PEP (Kiapp 0.54 mM); inhibition by ATP was not reversed by L‐alanine. Experiments employing BMA and MPA with gill tissue pieces subjected to hyperosmotic stress indicated that the PEPCK reaction in gill tissue was not significantly involved in the regulation of carbon flow for the synthesis of L‐alanine as an intracellular osmotic buffer.
Regulation of parthenogenetic activation of metaphase II mouse oocytes by pyruvate
Authors: Kim Haekwon , Schuetz Allen W.
Published: June 2005

We report that parthenogenetic activation (pronuclear formation) is induced during in vitro culture of recently ovulated (13–14 hr post‐hCG) mouse oocytes in pyruvate deficient medium. Pronuclear formation occurred when oocytes were cultured in medium containing 1/10X (Pyr−) or lower concentrations of pyruvate but failed to occur either in oocytes cultured in the presence of 0.47 mM (1X, Pyr+) or 1/2X pyruvate or in oocytes cultured in the absence of pyruvate but with cumulus cells. Pronuclear formation was evident within 8 hr of culture and completed by 16 hr and remained intact during continuous culture in Pyr− medium. Transfer of pronuclear oocytes to Pyr+ medium resulted in pronuclear membrane disassembly and further parthenogenetic development. A similar incidence of parthenogenetic activation occurred when recently ovulated oocytes were cultured in the presence of cycloheximide but not following ethanol or hyaluronidase treatment. However, both ethanol and hyaluronidase induced pronuclear formation in in vivo aged oocytes. Results suggest that the type of activation induced varies with the age of the oocyte and the nature of the stimulus. Amino acid uptake ([35S]methionine) by oocytes was unaffected by Pyr− culture whereas incorporation into protein was markedly inhibited. Gel electrophoretic analysis of labeled egg extracts revealed a marked inhibition of egg protein synthesis after 4 hr of culture in Pyr−. The occurrence of a cortical reaction was monitored by binding of fluorescent labeled lectin to the oocyte surface. A cortical reaction occurred in response to ethanol treatment of freshly ovulated and in vivo aged oocytes cultured in Pyr+ medium but not in pronucleate oocytes induced by Pyr− culture. Suppression of ethanol‐induced cortical reaction by Pyr− culture was restored following transfer of oocytes to Pyr+ medium. Results demonstrate that nuclear events as well as plasma membrane events can be simply regulated by controlling the amount of energy substrate available to the germ cell. Effects of Pyr− culture in inducing pronuclear formation appear to be mediated in a large part via inhibition of protein synthesis.
Growth of mouse embryos implanted in the subcutaneous tissue of recipient mice
Authors: Bevilacqua Estela Maris A. F. , Abrahamsohn Paulo A.
Published: June 2005

Eight‐days‐old mouse embryos were transferred to the subcutaneous tissue of the dorsal skin of host mice. A high rate of embryos developed into hemorrhagic (HN) or nonhemorrhagic nodules (NHN). The latter had trophoblastic cells as well as embryoblastic derivatives whereas HN contained almost only trophoblastic cells.

At least two kinds of trophoblastic cells were present in NHN: small cells and large cells similar to trophoblastic giant cells. In HN most trophoblastic cells arranged themselves into a network whose meshes contained host blood. Although embryoblastic derivatives such as endoderm and Reichert's membrane were apposed to host connective tissue cells, trophoblastic cells were always surrounded by collagen fibrils or by a layer of an amorphous material.
Plasma levels of glucose, ketone bodies, lactate, and alanine in the vascular supply to and from the brain of the adult American bullfrog (Rana catesbeiana)
Authors: Gibbs Scott R. , deRoos Roger M.
Published: June 2005

Serial, paired blood samples were collected via cannulae chronically placed in the common carotid artery (A) to and the internal jugular vein (V) from the brain of the fasted adult American bullfrog (Rana catesbeiana). Plasma glucose, β‐hydroxybutyrate, acetoacetate, lactate, and alanine levels were measured by standard enzymatic procedures. Cannula failure ended sampling after 1–2 days in most animals. The common carotid artery plasma metabolite levels were greatest at the time of surgery and subsequently declined to relatively stable levels. The summarized data indicated glucose uptake and alanine release by the brain, but no significant β‐hydroxybutyrate or lactate A‐V percentage changes. Initially, acetoacetate levels also were measured, but were discontinued in favor of continued β‐hydroxybutyrate determinations when no significant A‐V percentage changes occurred. Separate analysis of the metabolite levels during the surgery and recovery period (⩽ 24 hr) and the “normal” under the experimental conditions period (> 24 hr) revealed that summarizing the data masked important A‐V percentage changes during the two different physiological conditions. Glucose was the only metabolite extracted by the brain during the ⩽ 24 hr period of elevated and subsequently declining metabolite levels. In contrast, glucose uptake did not occur during the > 24 hr period of stable levels, but there was lactate release.

If the bullfrog brain stores substantial glycogen as do the other ectothermic vertebrates studied, glucose uptake when plasma levels are elevated, for example after feeding, may serve both to fuel the brain and to replenish endogenous glycogen reserves that may be mobilized to provide glucose for the brain after plasma glucose levels return to normal. Assuming that mammalian and bullfrog metabolic pathways are the same, the release of lactate and alanine by the brain, possibly to remove excess pyruvate and to regenerate NAD+, is consistent with this hypothesis. It remains to be determined for how long endogenous energy sources alone can support the bullfrog brain, and if plasma glucose, ketones, and/or other energy sources are extracted as endogenous brain fuels become exhausted.
Subcellular localization of two glutamine‐dependent carbamoyl‐phosphate synthetases and related enzymes in liver of Micropterus salmoides (largemouth bass) and properties of isolated liver mitochondria: Comparative relationship with elasmobranchs
Authors: Cao Xueying , Kemp Jon R. , Anderson Paul M.
Published: June 2005

Two different glutamine‐dependent carbamoyl‐phosphate synthetase (CPSase) activities are preset in tissues of Squalus acanthias (spiny dogfish shark, a representative elasmobranch) (Anderson, Biochem. J. 261:523–529, 1989). CPSase III (acetylglutamate‐dependent) activity is present in liver mitochondria; the function of this enzyme is related to urea synthesis for the purpose of osmoregulation, and the properties are analogous to the classical ammonia and acetylglutamate‐dependent CPSase I in liver mitochondria of ureotelic species (except that the nitrogen‐donating substrate is glutamine). CPSase II activity is present in spleen and other extrahepatic tissues, but is apparently absent from liver; the function of this enzyme is related to pyrimidine nucleotide biosynthesis, and the properties are analogous to mammalian CPSase II activities (independent of acetylglutamate, inhibited by UTP, and activated by 5‐phosphoribosyl 1‐pyrophosphate). Glutamine synthetase in shark liver is localized in the mitochondria, but the spleen glutamine synthetase is localized in the cytosol. Liver of largemouth bass (Micropterus salmoides, a freshwater teleost) also has CPSase III activity; however, unlike shark, aspartate carbamoyltransferase activity is also present in bass liver. Consequently, a major objective of this study was to establish if two different glutamine‐dependent CPSase activities are present in a single tissue (liver). Two different glutamine‐dependent CPSase activites were found to be present in bass liver, a mitochondrial CPSase III and a cytosolic CPSase II. The CPSase II activity, in contrast to the shark spleen CPSase II, appears to be associated with aspartate carbamoyltransferase and dihydro‐orotase activities, as found in mammalian species. As in shark, ornithine carbamoyltransferase, arginase, and phosphoenolpyruvate carboxykinase activities in bass liver are localized in the mitochondria, but, in contrast to shark, glutamine synthetase activity is localized in the cytosol. The properties of isolated, actively respiring bass liver mitochondria were investigated for comparison with previously reported properties of shark liver mitochondria. Respiration of bass mitochondria is supported by succinate and glutamate plus malate, but not by β‐hydroxybutyrate, palmitoyl‐CoA, citrate, or glutamine, all of which serve as excellent substrates for dogfish shark liver mitochondria. In contrast to shark mitochondria, bass mitochondria are not capable of ketone body formation and, of particular interest, do not catalyze citrulline formation from glutamine. CPSase III activity was not present in two other species of teleost fishes closely related to largemouth bass. The occurrence and function of CPSase III in teleost fishes are discussed.
Development and DNA polymerase activities in cultured preimplantation mouse embryos: Comparison with embryos developed in vivo
Authors: Kiessling Ann A. , Davis Howard W. , Williams Catherine S. , Sauter Ronald W. , Harrison Linda W.
Published: June 2005

Embryos from supervulated female mice that developed in vitro from the twocell stage were compared with in vivo embryos with respect to yield of blastocysts, number and types of cells, morphology in histologic section, and DNA polymerase activities. Significantly more embryos developed into blastocysts in vitro (93%) than in vivo (18%). Inner cell mass (ICM) cells comprised approximately 30% of total cells in late morula/early blastocyst stage embryos developed either in vitro or in vivo. However, the in vitro embryos developed approximately half the number of total cells as in vivo embryos, did not develop endoderm, and did not develop abembryonic trophoblast cells with morphologic characteristics of late preimplantation in vivo embryos. DNA‐dependent DNA polymerase activities in in vitro embryos decreased in correspondence with the decrease in cell number resulting in per cell levels comparable to in vivo embryos. In contrast, the poly (A)·oligo(dT)‐dependent DNA polymerase activity was the same in embryos developing either in vitro or in vivo, indicating different regulatory mechanisms for the two enzyme activities. A variety of nutrients and growth factors in the culture medium did not increase cell numbers or DNA polymerase activities in embryos cultured for 3 days; extending the culture an additional 24 hours resulted in a loss of ICM cells and decreases in both DNA polymerase activities. These results show that the retarded growth of embryos in vitro is equally distributed between ICM and trophoblast, is not reversed by culture conditions that include serum growth factors, and is not due to decreased cellular levels of DNA polymerase activities.
Storage and release of neuropeptides from the corpus cardiacum of the Eastern lubber grasshopper, Romalea microptera
Authors: Spring Jeffrey H. , Gäde Gerd
Published: June 2005

In this study, we describe a method for the separation of AKH/RPCH‐family peptides using isocratic high performance liquid chromatography (HPLC). This improved method is simple, rapid, and sensitive and bypasses many of the difficulties associated with gradient HPLC. We have previously shown that the lubber grasshopper, Romalea, accumulates large quantities of the native peptides Ro I and Ro II (Spring and Gäde, Journal of Experimental Zoology 241:41–50, 1987). In the present study, we used isocratic HPLC to follow the changes in the amount of Ro I and Ro II during development. Overall the amount of neuropeptide found in the corpora cardiaca (CC) increases 34‐fold from larval instar III to late adult, with the greatest quantitative increase (3,000 pmol total in males) occurring during the 6 weeks of adult life. The Ro I: Ro II ratio also changes during development, from 5.4 in larval instar III to 2.0 in late instar V and finally to 4.0 in mature adults. Under physiological conditions these neuropeptides can be released from Romalea CC isolated in vitro, when the CC are depolarized with high‐potassium saline. This release is calcium‐dependent and 10% to 15% of the total peptide stores are mobilized during a 1‐hour treatment with high‐potassium, plus calcium, saline. More than one‐third of the mobilized peptide is released within the first 5 min. In locusts, release of the AKH peptides is reported to be mediated by the neurotransmitter octopamine, acting via the adenylate cyclase pathway. In Romalea, however, we find no evidence for either octopamine or cAMP‐mediated release of neuropeptide.
Neomycin, an inhibitor of phosphoinositide hydrolysis, inhibits the resumption of bovine oocyte spontaneous meiotic maturation
Authors: Homa Sheryl T.
Published: June 2005

The possibility that the intracellular signals generated upon phosphoinositide hydrolysis are involved in regulating bovine oocyte spontaneous meiotic resumption was investigated. Oocytes were mass‐harvested and cultured in 2A‐BMOC medium supplemented with 0.5% bovine serum albumin in the presence or absence of neomycin (an inhibitor of phosphoinositide hydrolysis) or phorbol myristate acetate (an activator of protein kinase C). The role of intracellular calcium was examined by preloading with BAPTA/AM (a calcium chelator) prior to culture. Meiotic maturation was scored cytogenetically. 1) Neomycin induces an irreversible inhibition of germinal vesicle breakdown which does not exceed 60% and is apparent at concentrations of 5 mM or above. Progression of meiosis past metaphase I is inhibited at concentrations of 2.5 mM or above. The full effect of neomycin is only apparent if it is presented to the oocytes within 3 h of follicular release, although germinal vesicle breakdown is not observed until 9 h culture under control conditions. 2) PMA alone has negligible effect on germinal vesicle breakdown, but it acts synergistically with 2 mM IBMX to inhibit this process. PMA has a dual effect on the progression of meiosis past metaphase I: 1 nM PMA has a stimulatory effect while 1 μM PMA blocks the ability of oocytes to reach anaphase I or beyond. These observations are not found with a non‐tumor‐promoting phorbol ester. 3) Spontaneous meiotic resumption is not significantly affected in the absence of added exogenous calcium. However, oocytes preloaded with BAPTA/AM exhibit a dose‐dependent inhibition of germinal vesicle breakdown, even in the presence of extracellular calcium. These results provide indirect evidence for a role for phosphoinositide metabolism in bovine oocyte maturation; it is suggested that intracellular calcium mobilization is prerequisite for meiotic resumption and protein kinase C may modulate cAMP‐dependent inhibition of this response.
Hydrodynamics of the feet of fish‐catching bats: Influence of the water surface on drag and morphological design
Authors: Fish Frank E. , Blood Brad R. , Clark Brian D.
Published: June 2005

The bats, Noctilio leporinus and Pizonyx vivesi, display similar hind foot morphologies specialized for their fish‐catching habits. The hydrodynamic drag force of isolated hind feet from fishing and closely related non‐fishing bats was measured in a water channel at velocities up to 0.74 ms−1. Drag force increased curvilinearly with increasing water velocity. The highest values of drag force for a given velocity were measured for the fishing bats, which have hind feet 1.8–3.9 times larger than related non‐fishing bats. However at high Froude Numbers, the dimensionless drag coefficient (CD) was 23–39% lower for fishing bats compared to non‐fishing bats. Cross‐sections of the laterally compressed toes of N. leporinus and P. vivesi conformed to hydrofoil designs with minimum drag capabilities, indicated by Fineness Ratio and shoulder position. Moveing at high Froude Numbers at the water surface results in the addition of spray drag to the other components of the total drag force (i.e., frictional, pressure, and wave). Spray drag is suppressed by a foil shape with a long pointed leading edge with a short rounded trailing edge. The configuration of the fishing bat toe with a sharp leading edge and relatively long forebody region compared to the maximum thickness provided a measurable drag reduction at the air‐water interface. It was concluded that hydrodynamic drag considerations at the water surface were important in the design of hind feed for fish‐catching bats.
Isolation of Raja erinacea basolateral liver plasma membranes: Characterization of lipid composition and fluidity
Authors: Smith Daniel J. , Ploch Stephen A.
Published: June 2005

Developing a method for isolating skate (Raja erinacea) basolateral liver plasma membranes, as well as characterizing the lipid composition and fluidity of these membranes, was the primary purpose of this study. Membranes were isolated using self‐generating Percoll gradients. Marker enzyme studies indicate that this preparation is highly enriched in the basolateral domain of the liver plasma membrane and largely free of contamination by intracellular organelles or canalicular membranes. Further, these membranes contain the agency responsible for Na+‐dependent alanine transport. This finding indicates that this membrane prepration will be useful for the study of skate liver plasma membrane transport processes.

The lipid composition and fluidity (as assessed by the fluorescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatriene) of the skate basolateral liver plasma membrane shows little variation among preparations. Further, DPH anisotropy plotted as a function of temperature yields a straight line (r = 0.99) which indicates that there is no lipid phase change in these membranes from 4° to 37°C. The membrane preparation does contain substantial phospholipase A2 activity. The function of this enzyme is, in part, to modify membrane lipid composition and fluidity in response to temperature variations; therefore, this finding suggests that in situ lipid metabolizing enzymes may play a central role in the adaptation of skate basolateral liver plasma membranes to changes in the ambient temperature.
Linkage analysis of the murine Hyal‐1 locus on chromosome 9
Authors: De Maeyer‐Guignard Jaqueline , Cachard‐Thomas Annie , De Maeyer Edward
Published: June 2005

We have recently described a new locus, Hyal‐1, which determines hyaluronidase variants in mouse serum. On the basis of segregation in recombinant inbred and congeric strains, Hyal‐1 was tentatively assigned to chromosome 9 (Fiszer‐Szafarz and De Maeyer, '89). In the present study we have performed a linkage analysis of Hyal‐1 using 156 backcross progeny of an interspecies cross of laboratory mice and Mus Spretus. Linkage was tested to two anchor loci on chromosome 9: d (dilute, a coat color locus) and Bgl‐s (a locus controlling β‐galactosidase activity). The gene order (from centromere) with intervening percentage recombination is d‐16.6 ( ± 2.9)‐Hyal‐1‐10.9 ( ± 2.4)‐Bgl‐s, indicating close linkage to H‐7 and Fv‐2.
Granulosa cell–oocyte interactions: The phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells
Authors: Cecconi S. , Tatone C. , Buccione R. , Mangia F. , Colonna R.
Published: June 2005

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle‐enclosed growing oocytes but not in cumulus cell‐enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle‐stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.
Partial purification of Xenopus laevis egg extract factor(s) that induce swelling in permeabilized human sperm
Authors: Brown David B. , Miskimins W. Keith , Ruddle Frank H.
Published: June 2005

A combination of adsorption (protamine‐agarose) and gel filtration (Sephacryl S‐300) chromatography was used to enrich for factor(s) in Xenopus laevis frog egg extract that induce nuclear swelling‐chromatin decondensation in permeabilized human sperm. It was determined that a 70‐fold purification of the factor(s) that induce human sperm nuclear swelling has resulted from the purification scheme. The reduced, active factor(s) have a Kav of 0.12 and an approximate molecular weight of 290,000 daltons. The extract nuclear swelling activity is sensitive to temmperaturs of 50°C and above, as well as proteolytic treatment. RNase and cycloheximide treatments of the extracts have no effect on the nuclear swelling activity. These data suggest that the egg extract factor(s) that induce nuclear swelling in permeabilized human sperm are protein(s) that are present in the unfertilized frog egg.
Changes in intercellular contacts, motility, and adhesiveness of the presumptive mesodermal cells of Bufo vulgaris embryos before and during gastrulation
Authors: Komazaki Shinji
Published: June 2005

The morphology and adhesiveness of the presumptive mesodermal cells (PMCs) of Bufo vulgaris embryos were examined before and during gastrulation. Before the late‐blastula stage, the PMCs were in close contact with one another and were polyhedral in shape. Intercellular contacts became loose and cells were round in shape at the late‐blastula stage. At the initial stage of gastrulation, the PMCs began to form hyaline blebs. The blebbing cells appeared to crowd in one region (the bleb‐forming region [BFR]), located in the deep marginal mesoderm: The blebbing cells decreased in number and changed into filopodia‐ and lamellipodia‐forming cells with the start of the migration of PMCs at the early‐gastrula stage.

Cells in the BFR and in the presumptive BFR were isolated, and adhesiveness of cells to fibronectin was examined. The results showed that the BFR cells begin to adhere to fibronectin at the early‐gastrula stage when the PMCs start to migrate along the inner surface of the blastocoelic wall.

A series of changes (i.e., loosening of intercellular contacts, the formation of hyaline blebs, and the development of the ability to adhere to fibronectin) is thus evident in the PMCs before and during gastrulation. These serial changes are discussed within the context of the way in which PMCs start to migrate at the early‐gastrula stage.
Expression of epidermal growth factor (EGF) and the EGF receptor in human tissues
Authors: Fukuyama Ryuichi , Shimizu Nobuyoshi
Published: June 2005

The EGF‐EGF receptor system has been widely examined for signal transduction, control of cell growth and differentiation, and in vivo physiological function and carcinogenesis. The localization of EGF receptors in vivo led to the idea that the system is operative in proliferation and differentiation of cells and tissues. However, a consensus for its distribution and function in human tissues has not yet been determined because of discrepancies in the reported results. Using a highly specific monoclonal antibody against the EGF receptor, we examined various tissues of an infant and adults as well as embryonal carcinoma. We observed restricted localization of EGF receptors in basal cells of epithelial tissues and duct cells of secretory tissues. Fibroblasts express a high level of EGF receptors when they are rapidly growing. Using the monoclonal anti‐EGF antibodies, we observed that EGF is localized to differential cells rather than to stem cells such as glandular tissues. We also observed that some cells express both EGF and the EGF receptor. All histochemical results indicated that in epidermis and various glandular tissues, EGF may be expressed in differentiating cells derived from the stem cells expressing EGF receptors.
Effects of oxygen concentration and clutch on sex determination and physiology in red‐eared slider turtles (Trachemys scripta)
Authors: Etchberger Cory R. , Phillips John B. , Ewert Michael A. , Nelson Craig E. , Prange Henry D.
Published: June 2005

In many species of reptiles, sex is determined by the incubation temperature of the egg. In the red‐eared slider turtle (Trachemys scripta), warm incubation temperatures produce females, cool ones produce males, and a narrow range of intermediate temperatures produces both sexes. The mechanism of sex determination has not been established. Some investigators have postulated that both total incubation time and developmental rate during the first third of development are better predictors of sex than is incubation temperature. Here we consider whether various oxygen concentrations might influence sex determination by altering total incubation time. We incubated T. Scripta eggs at various oxygen concentrations, and found that while total incubation times were significantly influenced by oxygen, sex ratios were not. Very low oxygen concentrations inhibited survival to hatching, In addition, total incubation time and the number of days to internalize the yolk after hatching were significantly lengthened by lowered oxygen concentrations. There were also strong effects of clutch on sex determination and physiology, but these effects were apparently not mediated by an effect on total incubation time.
Purification and characterization of an acidic β‐galactosidase from the hepatopancreas of the shrimp Penaeus japonicus (Crustacea: Decapoda)
Authors: Chuang Nin‐Nin , Yang Bei‐Chia , Yeh Che‐Chung
Published: June 2005

An acidic β‐galactosidase was purified approximately 2,500‐fold to homogeneity from the shrimp Penaeus japonicus. with a final specific activity of 4,000 units/mg of protein. SDS‐polyacrylamide gel electrophoresis revealed the monomers of the acidic β‐galactosidase to have a relative mass of 66,000. Since the active acidic β‐galactosidase was found to have a relative mass of 140,000 by Ferguson plot analysis, the purified enzyme was concluded to be dimeric. No protective polypeptide was identified in the final preparation of purified β‐galactosidase. The shrimp enzyme was found to be thermolabile. Upon heating at 56°C for 10 min, the enzyme lost 50% of its activity. The acidic β‐galactosidase had an isoelectric point (pI) of 4.6 ± 0.1 and the enzyme was sialyated. The shrimp enzyme had a pH optimum of 5.5 and its Km was 43 μM with 4‐methylumbelliferyl‐β‐D‐galactoside as substrate. The enzyme was inhibited by both Mn++ and Zn++ ions.
Metabolism of progestogens during in vitro meiotic maturation of follicle‐enclosed oocytes of the goldfish (Carassius auratus)
Authors: Lessman Charles A.
Published: June 2005

Progestogens are known to elicit meiotic maturation of fish oocytes in vitro. Exogenous 17α‐hydroxyprogesterone (HP), as assessed by radioimmunoassay (RIA), decreased exponentially to less than 10% of the initial level in media during a 48 hr incubation with large (i.e., 1 mm dia.) ovarian follicles of goldfish undergoing meiotic maturation in vitro. During follicle incubation, media, into which [3H]‐HP was introduced, showed exponential showed exponential reduction of [3H]‐HP with a concomitant increase in the number of [3H]‐HP metabolites. Analysis of follicle extracts revealed that small amounts of [3H]‐HP were taken up by the follicles early in the incubation (i.e., 3 hr) and converted to several metabolites; the major one of these, which accumulated in the follicle, comigrated with 17α, 20β dihydroxy 4‐pregnen 3‐one (DHP). Autoradiography of extracts run on TLC revealed an increasing number of metabolites with time, i.e., 9 major spots for media, 6 major spots for follicles incubated 24 hr with [3H]‐HP. Three of the more prominent metabolites, DHP, 3α, 17α‐dihydroxy5β‐pregnan 20‐one, and 5β‐pregnan3α, 17α, 20β‐triol, were identified tentatively by comigration with standards on TLC and crystallization to constant specific activity. HPLC of extracts revealed a selective accumulation of DHP in follicles over time i.e., this steroid was the major metabolite peak associated with follicles by 24 hr incubation. Nevertheless, the process of oocyte maturation had already been initiated before the peak level of DHP had been reached. By contrast, HP diminished with time in follicles despite the relatively high levels occurring in the media. This research indicates that a complex steroid milieu at the level of individual, fully grown, ovarian follicles is produced in vitro from exogenous HP; significant HP metabolism occurs by 6 hr incubation, well before the process of oocyte maturation (i.e., germinal vesicle migration and dissolution) has begun.
Identification and expression of boar sperm proteins in the reproductive tract that interact with antibodies in serum from an infertile woman
Authors: Haneji Tatsuji , Nagano Toshio , Koide S. S.
Published: June 2005

Serum designated as IS obtained from a young healthy infertile woman induced a head‐to‐head agglutination of ejaculated boar sperm. The immunoglobulin G (IgG) prepared from IS localized to the acrosomal region of the sperm head obtained from the corpus and cauda epididymis as determined by an indirect immunofluorescent method. The IgG interacted with a boar sperm protein with an estimated molecular weight of 45‐kDa, determined by sodium dodecyl sulfate‐polyacrylamide gel electrophoretic (SDS‐PAGE) immunoblotting technique. However, the IgG did not interact with proteins extracted from sperm obtained from the testis and caput epididymis or from non‐gonadal tissues including liver, kidney, spleen, muscle and serum. The IgG interacted with additional proteins of about 75‐and 38‐kDa present in the corpus and cauda epididymal fluids but not those in the caput epidiymal fluid. The staining intensity of the 75‐kDa band was reduced and that of the 38‐kDa was nullified with ejaculated seminal plasma proteins. The interacting proteins were adsorbed when chromatographed on Concanavalin A Sepharose column, suggesting that they are glycoproteins.
Proteolytic activity in the fat body during the pupal‐adult metamorphosis of the silkworm, Bombyx mori
Authors: Yaginuma Toshinobu , Ushizima Miwa
Published: June 2005

Proteolytic activity with an optimum pH of 4.0 was found in fat bodies of 5 to 6 day‐old female pupae of the silkworm, Bombyx mori, using azocoll as a synthetic substrate. The activity was strongly inhibited by p‐chloromercuric benzoate at 0.5 mM, although inhibitory effect of several kinds of compounds on proteolytic activity was examined. Developmental changes in the activity were associated with two peaks of proteolytic activity related to the larval‐pupal and pupal‐adult transformation, the activity in the fat body of females was clearly higher than that of males. The profile of the proteolytic activity after chromatography on a column of Sephacryl S‐300 showed that, in the female fat body, the highest activity was in the fraction with a molecular weight of about 232,000 daltons while, in male fat body, the highest activity was in the fraction with a molecular weight of about 105,000 daltons. The storage proteins were digested when extracts from fat bodies, which contain storage proteins and proteases, were incubated and only when the pH of the extract was adjusted to 4.0. In the case of female fat body, storage protein 1 was degraded earlier than storage protein 2. Furthermore, the administration of 20‐hydroxyecdysone (5 μg/animal) to the isolated abdomens of female pupae (dauer‐pupae) induced the proteolytic activity in the fat body in parallel with ovarian development. These results suggest that the proteases in female and male fat bodies are responsible for the utilization of storage proteins during pupal‐adult development.
Comparative study of orb web hygroscopicity and adhesive spiral composition in three araneid spiders
Authors: Townley Mark A. , Bernstein David T. , Gallagher Kathleen S. , Tillinghast Edward K.
Published: June 2005

Orb web hygroscopicity was examined in three species of araneid spiders, Argiope aurantia, Argiope trifasciata, and Araneus cavaticus. Both whole webs and fractions generated from them (water‐soluble and ‐insoluble fractions; ethanol‐soluble and ‐insoluble subfractions [Etsol and ‐insol]) were examined at various relative humidities. In general, the water‐soluble fraction, consisting of low molecular weight components (LMW) of the adhesive spiral cover, was more hygroscopic than the water‐insoluble (protein) fraction. As the Et‐sol of the water extract was typically more hygroscopic than the Et‐insol, it appears that organic LMW are more responsible for water adsorption by the adhesive spiral cover than inorgnic LMW. Hygroscopicity measurements made on individual synthetic or commercially obtained LMW compounds known to be in the web (both organic and inorganic) agree with this interpretation.

As a first attempt toward correlating web hygroscopicity with composition, the water‐soluble web fractions from the three species were examined by proton nuclear magnetic resonance (1H‐NMR) spectroscopy and the molar percentages of the organic LMW were estimated. To accomplish this, it was necessary to identify the principal organic LMW in the web of A. trifasciata (since, unlike the situation in other araneids which have been studied, GABamide is not the principal organic LMW in A. trifasciata). Using 1H‐NMR and thin layer chromatography this compound was identified as N‐acetylputrescine. While considerable interspecific variation in the molar percentages of GABamide, N‐acetylputrescine, isethionic acid, and N‐acetyltaurine was observed, interspecific differences in Et‐sol hygroscopicities were not apparent.