bluuebunny/crossref_metadata_2025 huggingface dataset:
The synthesis and localization of glycosaminoglycans in striated muscle differentiating in cell culture
Authors: Ahrens Patricia Buckley , Solursh Michael , Meier Stephen
Published: June 2005

Day 3 mass muscle cultures incorporated 35SO4= and 3H· glucosamine into specific glycosaminoglycans that were identified by a variety of methods (enzyme susceptibility, nitrous acid degradation, electrophoresis, thin layer chromatography). More than 75% of the newly synthesized glycosaminoglycans appeared in the medium. In the medium, the glycosaminoglycans consisted of primarily hyaluronic acid (∼35%) and chondroitin sulfate (∼40%) with smaller amounts of chondroitin (∼15%) and heparan sulfate (∼10%). Among the glycosaminoglycans that remained in the cell layer, chondroitin sulfate (∼45%) and heparan sulfate (20‐40%) predominated and only a small amount of chondroitin (∼15%) and hyaluronic acid (∼5%) were present. In both the medium and cell layer, greater than 80% of the chondroitin sulfate was chondroitin‐6‐sulfate. Although mass cultures contain some non‐muscle cell types which undoubtedly contributed to total glycosaminoglycan synthesis, susceptibility of the autoradiographic grains associated with cells in muscle clones to testicular hyaluronidase digestion suggests that at least some glycosaminoglycans (chondroitin sulfate, in particular) were synthesized by muscle cells.

Transmission electron microscopy revealed the presence of ruthenium red staining globular units along the surfaces of myotubes. Destruction of the integrity of surface associated ruthenium red stained material by both Streptomyces hyaluronidase and testicular hyaluronidase suggests that both hyaluronic acid and chondroitin sulfate are present on muscle cell surfaces.
Characterization of the divalent cation activated adenosine triphosphatase from the membrane of the cardiac cell of Modiolus demissus demissus
Authors: Watts John A. , Pierce Sidney K.
Published: June 2005

Cell‐surface membranes from Modiolus demissus possess diva‐lent cation stimulated adenosine triphosphatase (ATPase) activity. The ATPase activity was stimulated by Ca2+ and to a lesser extend by Mg2+ in the presence of 3 mM ATP. When Ca2+ and Mg2+ were combined in the sea‐water ratio of 1:5, constant ATPase activity, intermediate between that in the presence of Ca2+ or Mg2+ alone, was observed from 10:50 mM to 1:5 mM Ca2+ Mg2+ The maximum rate of substrate hydrolysis occurred at pH 7.5–7.7, 37–45°C and with 3–5 mM ATP. Other triphosphate substrates were cleaved but ADP, AMP or p‐nitro‐phenylphosphate were not, showing that the enzyme is a triphosphatase Mg2+ ATPase activity was not inhibited by ouabain or ruthenium red, and only slightly inhibited by ethacrynic acid, while La3+ had a slight stimulatory effect.
Organ culture of mammalian skin and the effects of ultraviolet light and testosterone on melanocyte morphology and function
Authors: Glimcher Mark Edward , Garcia Raul I. , Szabó George
Published: June 2005

Scrotal skin of black Long‐Evans rats and human thigh skin were maintained in vitro as organ cultures for as long as 14 days, and examined histologically using the combined skin splitting and Dopa techniques. Selected rat skin cultures received testosterone in the culture medium and/or were irradiated with ultraviolet light (290–320 nm UVL). With increased time in culture, scrotal melanocytes round up and there is an increase in epidermal pigmentation. Human skin behaves similarly; after eight days in vitro human melanocytes also become rounded, but remain strongly Dopa‐positive. Addition of exogenous testosterone to cultured rat skin maintains dendritic morphology of melanocytes, but cell body size is still reduced. UVL irradiation stimulates melanocytes in rat skin cultures, maintaining their dendritic morphology and increasing epidermal and dermal pigmentation. Cultured skin receiving both UVL and testosterone illustrates a synergistic effect. Electron microscopic examination of cultured rat skin shows the presence of large melanosome complexes in keratinocytes, much larger than those found in vivo. Melanocytes appear to be active as they contain an extensive Golgi zone, rough endoplasmic reticulum, and melanosomes in various stages of formation. Dermis contained many dermal melanocytes and macrophages laden with melanosomes, correlating with the increased visible dermal pigmentation in vitro.

This UVL stimulation of melanocytes in our skin organ cultures contrasts with the lack of melanogenic stimulation found in melanoma cell cultures. Our findings suggest that the intact epidermal melanin unit may be necessary for UVL stimulation of melanocytes.
Calcium redistribution in individual cells correlated with ionophore action on motility
Authors: Babcock Donner F. , Stamerjohn David M. , Hutchinson Thomas
Published: June 2005

Intact bovine epididymal spermatozoa were rapidly frozen (but otherwise unfixed) and examined in the cryogenic stage of a scanning electron microscope equipped with an energy‐dispersive x‐ray analysis system. Distributions of calcium (also of P, S, K and Cl) were determined in 0.1 μm2 areas from the acrosome, nucleus, midpiece and flagella of cells that were subjected to a variety of conditions experimental that altered sperm total calcium content.

Spectrophotometric measurements using the metallochromic indicator, murexide, showed that a net accumulation of calcium followed in vitro incubation of sperm in the presence of this cation. Accumulated calcium was distributed homogenously amongst the cell population, but was apparently concentrated in the mitochondria contained within the sperm midpiece as determined by electron microanalysis of individual cells. A net loss of previously accumulated calcium followed treatment of sperm with low levels of the divalent cation ionophore, A23187. Electron microanalysis indicated a corresponding depletion of mitochondrial calcium content. In contrast, subsequent treatment of sperm with high levels of A23187 resulted in net calcium uptake as determined by murexide absorbance changes. Electron microanalysis indicated that this reaccumulated calcium was distributed throughout the intracellular space. No significant changes in the intracellular concentrations or distributions of other elements were observed following ionophore treatment. These results support previous suggestions that the mechanism of motility activation mediated by A23187 involves an action of increased intracellular Ca2 + concentrations upon the contractile apparatus of the sperm flagella.
Further evidence in support of a role for hamster sperm hydrolytic enzymes in the acrosome reaction
Authors: Lui Chung W. , Meizel Stanley
Published: June 2005

The effects of trypsin inhibitors and phospholipase inhibitors on the acrosome reaction of washed cauda epididymal sperm of golden hamsters were studied using two different incubation systems. One incubation system, a non‐synchronous acrosome reaction inducing system, included the use of a highly purified BSA and a protein‐free motility factor preparation from hamster adrenal gland. The other system was a relatively synchronous acrosome reaction‐inducing‐system utilizing the calcium ionophore A23187. Acrosome reactions were inhibited by three low molecular weight synthetic trypsin inhibitors, benzamidine, NPGB and TLCK, when they were added five minutes prior to the initial occurrence of acrosome reactions in the non‐synchronous system or five minutes prior to induction of acrosome reactions by A23187 in the synchronous system. Two phospholipase A inhibitors, p‐bromophenacyl bromide and mepacrine, were also effective in inhibiting hamster sperm acrosome reactions in both incubation systems. TPCK, an inhibitor of several non‐trypsin‐like proteases, indomethacin, a prostaglandin synthetase inhibitor, and soybean trypsin inhibitor, a large molecular weight polypeptide, did not inhibit acrosome reactions. The inhibition of those acrosome reactions induced by A23187 provides further indirect evidence that the effective inhibitors were functioning at a site within the sperm. The overall results provide: (1) further support for our earlier work suggesting the involvement of an internal trypsinlike enzyme (presumably acrosin) rather than an exogenous trypsin‐like enzyme in the hamster sperm acrosome reaction and (2) the first evidence suggesting the possibility that a sperm phospholipase may also be involved in the mammalian acrosome reaction.
Polymorphism, genetic stability, and autosomal location of trimeric nucleoside phosphorylase in Peromyscus eremicus cell lines
Authors: Maxwell Beth Lynn , Siciliano Michael J.
Published: June 2005

Nucleoside phosphorylase (NP: EC 2.4.2.1) has not been demonstrated to be an extensively polymorphic enzyme locus in mammals. We have studied NP electrophoretically in five independently derived cell lines established from Peromyscus eremicus as well as in various tissues of a sixth animal. Four different NP phenotypes involving three different alleles were resolved. The data suggest that (1) the enzyme is trimeric and its genetic locus is polymorphic in P. eremicus, (2) heterozygous enzyme phenotypes are stable during long‐term culture, and (3) the enzyme locus is autosomal in Peromyscus.
Physical factors and neurosecretion in the control of reproduction in Melampus (Mollusca: Pulmonata)
Authors: Price Christopher H.
Published: June 2005

Melampus bidentatus a saltmarsh snail living in the high littoral zone, lays eggs in phase with the moon, three to four times in early summer. Repetitive cycles of aggregation, egglaying, and dispersion were quantified in field studies. Physiological preparedness for breeding begins with an essential winter vernalization (<5°C). Breeding condition (growth of ovotestis, etc.) is induced in early spring only after both photoperiodic (>14 hours per 24 hours) and temperature (>15°C) thresholds are exceeded. Animals collected during winter could be induced to lay eggs if given spring‐like conditions; progressively shorter induction periods were effective as spring approached. Yearround histological studies of neurosecretory cells in the central ganglia of field animals showed staining patterns in two groups of neurons that were closely related to reproductive events. The medium cells (N =7) of the lateral lobe in each cerebral ganglion showed an accumulation of neurosecretory material (NSM) only in April, during the period when the natural photoperiod first exceeds 14 hours. These cells may mediate the light requirement and stimulate early reproductive events, such as ovotestis growth. The giant cells (N = 15) of the visceral ganglia stained heavily only during the reproductive season. They showed repetitive cycles of accumulation and subsequent depletion of NSM in phase with egg‐laying cycles. These neurons, signalled by some unknown but semilunar‐related cue, apparently synthesize and store NSM prior to each egglaying; release of this material may initiate the behavioral and physiological events of egg deposition.
Quantitative determination of titers of anti‐zona serum
Authors: Tsunoda Y. , Sugie T. , Mori J.
Published: June 2005

The titers of rabbit antiserum against isolated mouse zonae pellucidae were examined by several methods in connection with inhibitory effect on fertilization.

The titers determined by zona precipitate, zona dissolution, indirect immunofluorescence and in vitro fertilization tests were 24, 21 to 24, 27 and 24, respectively. Cytotoxic effect could not be detected from zona antibody. The indirect immunofluorescence was most sensitive for detection of zona antibody but did not represent the extent of inhibitory effect on fertilization. The titer obtained by zona precipitate test was most close to the titer obtained by inhibitory effect on in vitro fertilization.

The present study also demonstrated that at least 0.0375 ml of antiserum per female mouse, equivalent to 0.15–0.25% of body weight, was necessary for inhibition of fertilization in vivo by passive immunization with anti‐zona serum.
Evidence for the continuation of meiosis and spermiogenesis in in vitro cultures of spermatogenic cells from Xenopus laevis
Authors: Risley Michael S. , Eckhardt Ronald A.
Published: June 2005

Spermatogenic cells were obtained from Xenopus laevis testes by collagenase dissociation and subsequently separated into two fractions by density gradient centrifugation in Metrizamide and by unit gravity sedimentation in bovine serum albumin gradients. One fraction was highly enriched for primary spermatocytes while the other fraction was highly enriched for early‐mid stage spermatids. The cells from each fraction were cultured separately in modified DNOM medium for 23 days to determine the extent to which differentiation could be maintained in vitro. Differential cell counts revealed that early‐mid stage spermatids continues to differentiate in vitro through the stages of cytoplasmic shedding. Spermatocytes passed through both meiotic divisions throughout the 23 day period and produced spermatids which also differentiated through the cytoplasmic shedding stages. The results demonstrated that significant differentiative potential is retained by Xenopus spermatogenic cells cultured in vitro even after dissociation and cell separation procedurs.
Hormone‐induced pigment translocations in amphibian dermal iridophores, in vitro: Changes in cell shape
Authors: Butman Bryan T. , Obika M. , Tchen T. T. , Taylor John D.
Published: June 2005

Hormone‐induced pigment translocation studies were conducted at both the light and electron microscopic levels on cultured dermal iridophores from the Mexican leaf frog, Pachymedusa dacnicolor. Two distinct types of dermal iridophores were characterized which differed in (1) their in vivo locations, (2) their overall morphologies in vitro, (3) their responses to α‐MSH, ACTH, c‐AMP or theophylline, (4) their physical alterations of light, and (5) certain ultrastructural features. One iridophore (Type I) was found to be physiologically responsive to the above hormones or agents by a reversible retraction of cellular processes and a thickening of the cell body, an event which is inhibited by cytochalasin B. The other iridophore (Type II) appeared to be unresponsive. Type I iridophores contain cube‐like pigmentary organelles, refractosomes, while Type II iridophores contain larger, bar‐shaped refractosomes. In addition, both iridophore types contain 60 and 100 Å microfilaments as well as microtubules. By in large, microfilaments were found within microvilli, beneath and parallel to the plasma membrane and in the perinuclear region. Occasionally, bundles of 100 Å microfilaments were found between layers of refractosomes in Type I iridophores. These results are discussed in relation to hormone‐induced changes in cell shape.
Epigenetic formation of lactate dehydrogenase isozymes in the house mouse, Mus musculus
Authors: Yamamura Ken‐Ichi , Ogita Zen‐Ichi , Markert Clement L.
Published: June 2005

The origin of mouse lactate dehydrogenase (LDH) subbands was investigated by using our miniaturized polyacrylamide gel electrophoretic apparatus. Mouse LDH isozymes are generated by combinations of three types of A subunit, the primary type and two epigenetically modified forms. These are designated A1, A2, and A3 in the order of their electrophoretic mobilities towards the anode. The A1 subunit arises from the covalent binding of molecules of glutathione through disulfide bonds to the original subunit, A3. The A2 subunit arises from the covalent binding of molecules of cysteine through disulfide bonds to the A3 subunit. All isozymes can be explained as tetramers composed of the three kinds of A subunit (A1, A2, or A3) in combination with B subunits to yield a total of 35 isozymes. The kinetic properties of these subbands were also examined. There was no difference between A24 and A34 in the Km for pyruvate and for lactate. Thermostability at 56°C was greater for A34 than for A24. The activities of tetramers at the electrophoretic position of A3B1 and A4 in extracts containing all five isozymes were increased by treatment of the extracts with high concentrations of reduced glutathione or cysteine with the concomitant disappearance or decrease in activity of tetramers at the position of B4 and A3B1. These results suggest that, in the presence of reduced glutathione or cysteine, LDH isozymes containing the B subunit are first dissociated and then the A subunits are preferentially recombined.
Injection of sperm heads into immature rat oocytes
Authors: Thadani Vijay M.
Published: June 2005

When sperm heads are injected into rat oocytes at the germinal vesicle stage the sperm heads remain intact until the germinal vesicle breaks down. Then they decondense but do not form pronuclei. This observation agrees with the results reported for in vitro fertilization of immature oocytes from rats, mice and hamsters.
Pyruvate kinases of salmon: Purification and comparison with the isozymes from birds and mammals
Authors: Guderley Helga , Cardenas Janet M.
Published: June 2005

Pyruvate kinase occurs as two major forms in coho salmon; the type M isozyme occurs primarily in muscle and heart, but type K has a more generalized tissue distribution, in parallel with the type K isozyme in other vertebrate systems. In order to assess the evolutionary relationships among the fish, avian, and mammalian isozymes of pyruvate kinase, we have purified the two isozymes from fish, have examined some of their physical properties, and have studied their immunological relationships to theavian and mammalian isozymes. Salmon type K is at least partially inactivated by antibody to bovine type L pyruvate kinase as well as by antibodies produced against chicken, bovine, and salmon type M isozymes. Salmon type M pyruvate kinase, on the other hand, is not significantly cross‐reactive with the bovine type L isozyme, but is at least partially inactivated by antibodies produced against bovine or chicken type M isozymes. Mammalian type L pyruvate kinase is immunologically distinct from either mammalian type K or type M, but salmon type K has some structural features in common with all three mammalian isozymes. Thus, salmon fish type K pyruvate kinase could be similar to a primordial form that was antecedent to the three major differentiated isozymes of higher vertebrates.
In vivo and in vitro studies on the early embryonic lethal tail‐short (Ts) in the mouse
Authors: Paterson Hugh F.
Published: June 2005

An histological study reveals that embryos homozygous for the mutation tail‐short (Ts) become abnormal as morulae, although a small amount of cell division may continue to take place during the following day. Homozygotes can be identified at 3½ days post coitum (p.c.) by several criteria including low cell number, absence of cavitation, and weak cytoplasmic staining with haematoxylin. A similar pattern of developmental arrest is exhibited during culture, although an initial degreeof cavitation occurs more frequently.
Lens formation from pericorneal epidermis in the presence of the old lens in larval Xenopus laevis
Authors: Filoni Sergio , Bosco Luigi , Paglioni Nadia , Cioni Carla
Published: June 2005

The lens‐forming capacity of the pericorneal epidermis of Xenopus laevis larvae at stage 51 has been investigated. The results obtained show that, whether or not the lens is present, the pericorneal epidermis can form a lens when it is in direct contact with the vitreous chamber of the eye. It is concluded that the posterior pole of the lens either does not produce any inhibiting substances or else is unable effectively to remove the factor(s) contained in the vitreous chamber which are responsible for the lens‐forming transformations of the pericorneal epidermis.
Alterations in the levels of stem cells (CFU‐s) and plaque‐forming cells (PFC) in mice during chronic phenylhydrazine‐lnduced hemolytic anemia
Authors: Kozlov V. A. , Zhuravkin I. N. , Coleman R. M. , Rencricca N. J.
Published: June 2005

The levels of splenic and medullary pluripotent stem cells (CFU‐s) and splenic plaque‐forming cells (PFC) to sheep red blood cells (SRBC) were monitored in mice during the course of mild chronic phenylhydrazine (PHZ)‐induced hemolytic anemia. The levels of CFU‐s were significantly depressed in the bone marrow and elevated in the spleen throughout. These changes are most likely due to increased CFU‐s migration from marrow to spleen. In this regard, migration of CFU‐s from limb‐shielded marrow to thespleen of irradiated PHZ‐treated mice was demonstrated. Lastly, the numbers of splenic PFC to SRBC were significantly and constantly depressed throughout the course of hemolytic anemia and enhanced erythropoiesis. Stem cell competition between erythroidand lymphoid lines of differentiation may have contributed to the observed reduced immune response.
Effects of hormones on post‐implantation mouse embryos in vitro. I. Insulin
Authors: Fisher D. L.
Published: June 2005

Day 9 mouse embryos were cultured in 50% fetal calf serum and 50% Waymouth's medium supplemented with various amounts of insulin. Unmodified medium contained approximately 25% of the normal adult mouse blood insulin. The addition of 0.1 to 1.0 μg insulin/ml to Waymouth's medium was most beneficial to in vitro development. There was a statistically significant increase in protein synthesis, RNA content, glucose utilization, and heart rate, as well as a significant decrease in pericardial edema. DNA, synthesis was unchanged or slightly decreased. The optimal amount of added insulin (0.05 μg/ml) is much higher than the normal circulating levels in adult mice.
Prolonged survival of skin allografts in mallards
Authors: Mueller Nancy S.
Published: June 2005

Skin allografts were exchanged between 25 male and 25 female mallards (Anas platyrhynchos) within 2 days after hatching. Of the 42 ducks that survived and whose grafts healed, 14% rejected rapidly before the grafts became feathered, 20% rejected between 3–6 weeks, and one duck rejected when he was 10 weeks old. Twenty‐seven of the 42 ducks (64%) retained their grafts for at least 5 months. The result demonstrate that, in mallards, neither inbreeding nor any treatment of the host is necessary for prolonged survival of skin allografts made shortly after hatching.
Possible control for tubulin synthesis during early development of an insect
Authors: Jäckle Herbert
Published: June 2005

Tubulin represents a major protein component synthesized during early embryogenesis of the chironomid midge Smittia spec. Both α‐ and β‐tubulin were identified by immunoprecipitation and subsequent analysis by two‐dimensional gel electrophoresis using purified Drosophila microtubule proteins as a marker. The synthesis of tubulin in Smittia embryos was not localized within regions perpendicular to the longitudinal axis. The messenger RNA for both α‐ and β‐tubulin appeared to be maternal in origin (maternal mRNA). The in vivo ratio of [35S]‐methionine incorporation into α‐tubulin relative to β‐tubulin was 1:2.1, and constant at least until the end of blastoderm stage. In a rabbit reticulocyte lysate, tubulin synthesis was directed by total RNA, poly(A)‐containing RNA, and nonpoly(A)‐containing RNA extracted from eggs immediately after deposition. However, the in vitro ratio of [35S]‐methionine incorporation into α‐ and β‐tubulin was 1:1. The results suggest that tubulin is synthesized in the early embryo using maternal mRNA. Therefore, posttranscriptional rather than transcriptional processess seem to regulate the synthesis of α‐tubulin relative to β‐tubulin.
Comparative extraocular muscle blood flow
Authors: Wilcox Lloyd M. , Keough Ellen M. , Connolly Raymond J. , Hotte Clifford E.
Published: June 2005

This study was designed to quantitatively determine blood flow to the extraocular muscles (EOM) of animals showing a wide range of ocular motility. Blood‐flow values (ml/minute/gm) for EOM in primates (0.25 ± 0.03), dogs (0.33 ± 0.06), sheep (0.12 ± 0.01), and geese (0.12 ± 0.04) were determined using the Reference Sample Method with 15 ± 3 μm 103Ru microspheres and complete cardiovascular monitoring. Mammalian EOM blood flow was significantly higher than that determined for other skeletal muscle; however, EOM blood‐flow values for sheep were significantly lower than those for primates and dogs.

No significant blood‐flow differences were found between avian EOM and other skeletal muscle, reflecting the unique avian ocular anatomy. These data indicate that high blood flow is not a general property of EOM and suggest a relationship between functional ocular motility and blood flow.
Transport of exogenous substrate and cell volume regulation in bivalve molluscs
Authors: Crowe John H.
Published: June 2005

Based on published values for transport kinetics, it has been suggested that uptake of free amino acids from the environment could not play a significant role in altering the intracellular pool of free amino acids during hypersaline acclimation in euryhaline bivalves. A review of more recent findings indicates that this suggestion requires reexamination. It has also been suggested that extracellular sources of amino acids within the animals could be utilized in altering the intracellular pool. Available data having bearing on that hypothesis are reviewed.
On the timing and activation of glycogen catabolism in fertilized sea urchin eggs
Authors: Baginski Richard M.
Published: June 2005

Decreased glycogen radioactivity was followed as an indicator of glycogen utilization following activation of Strongylocentrotus purpuratus eggs. Within 15 minutes of exposure to sperm or ammonia, egg glycogen radioactivity decreased. The level of radioactivity in control (unfertilized) eggs remained relatively constant. These results suggest enhanced egg glycogenolysis is a pH‐sensitive event independent of the “respiratory burst”.
Significance of periodic fluctuations in the haemolymph carbohydrates of Scylla serrata (Forskal) during starvation and repeated injury
Authors: Saravanan T. S. , Ravindranath M. H.
Published: June 2005

Haemolymph carbohydrates of S. serrata, including reducing sugars (RS), nonreducing sugars (NRS), and glycogen (free and bound to protein) showed intermittent fluctuations in animals subjected to starvation in combination with injury and exsanguination stress (Group I), whereas in animals subjected to starvation alone (Group II) only RS levels showed such significant periodic fluctuations. The timing of periodic ‘peaks’ in the haemolymph total free sugar (TFS) and RS coincided in both groups of animals. Haemolymph TFS, although declining throughout 33 days of starvation, increased significantly on three different days. Similar patterns were observed in the patterns of reducing sugar. Levels of haemolymph NRS and glycogen were greater throughout the 33 days of starvation in group I than in group II. NRS, glycogen, and protein‐bound polysaccharides (PBS) exhibited significant fluctuations only in Group I animals. The significance of the periodic fluctuations in haemolymph carbohydrates is discussed.
Morphological observations on cleavage of the egg of the medaka, Oryzias latipes, following injection with flagellar microtubules of sea urchin spermatozoa
Authors: Ohta Tadayuki , Iwamatsu Takashi
Published: June 2005

Mature unfertilized eggs of the medaka, Oryzias latipes, were injected with sperm flagellar microtubules of the sea urchin, Hemicentrotus pulcherrimus, and were observed with reference to the process of early development. Groups of injected eggs, most of which initiated cleavage and developed to morulae, were fixed with Feulgen fixative at 10, 30, 45, 60, 80, and 90 minutes and at 5 hours after injection. Light microscopic observation revealed that by 45 to 60 minutes, the eggs had radially organized astral fibers originating from the periphery of the droplet of injected microtubule suspension. First cleavage then occurred at 80 to 90 minutes. At the morula stage (5 hours), each blastomere always contained an aster‐like structure composed mainly of endoplasmic reticula. These observations confirmed that the injected flagellar microtubules induced aster formation and first cleavage in unfertilized eggs of the fish.
Sperm‐induced cell cycle activities in blastomeres arrested by the cytostatic factor of unfertilized eggs in Rana pipiens
Authors: Shibuya Ellen K. , Masui Yoshio
Published: June 2005

Suspensions of lysolecithin‐treated sperm were injected into blastomeres of two‐cell embryos which had been arrested with the extract of unfertilized eggs containing cytostatic factor (CSF). Whereas the CSF‐arrested blastomeres remained unchanged if no sperm were injected, those injected with sperm formed abortive cleavage furrows. The injected sperm exhibited characteristics similar to those associated with sperm injected into activated eggs. These were nuclear decondensation to the pronucleus, DNA synthesis, and late chromosome condensation to the metaphase stage. The activity of CSF‐arrested blastomere cytoplasm to promote meiotic maturation when injected into ovarian oocytes rapidly decreased following sperm injection, but later fluctuated at low levels. It was suggested that the CSF‐arrested blastomeres retained the potential to reinitiate cell cycle activities which could be evoked by sperm injection.
Experimental evidence for effective heterokaryon formation in the ciliate Euplotes crassus
Authors: Miceli Cristina , Luporini Pierangelo
Published: June 2005

Morphological mutant doublets of Euplotes crassus containing two nuclear complexes and expressing a definite mating type were conjugated with normal singlets of a complementary mating type. After conjugation a class of clones was obtained each of which consisted of doublets having both a capability to form intraclonal pairs and no restriction in the mating spectrum. Doublets were isolated from this class of clones and were subsequently allowed to split into their component halves (singlets). The two singlet sib‐lines deriving by vegetative growth of a split doublet were tested for mating type and were usually found to be different. Also, different allelic combinations were consistently shown to exist at the mt locus, i. e., the locus determining the mating type. Our data support the hypothesis that these doublets which gave rise to two different singlet sib‐lines should have contained nuclear complexes with different, active mt alleles. Thus these doublets capable of expressing two mating types at the same time can be considered to be effective heterokaryons.
Cyclophosphamide‐induced leukopenia and suppression of limb regeneration in the adult newt, Notophthalmus viridescens
Authors: Schotté Oscar E. , Sicard Raymond E.
Published: June 2005

Cyclophosphamide (cytoxan), a clinical immunosuppressant, produced a marked, persistent leukopenia (> 40% reduction) in adult newts in contrast to a transient, milder leukopenia accompanying amputations without cytoxan treatments. In addition, cytoxan suppressed the formation of regeneration blastemata initially; however, blastema formation occurred if a second amputation was performed two or more weeks following the cessation of cytoxan treatments. The failure of the leukopenias of these latter cases to be corrected suggests that although a cytoxan‐induced leukopenia is correlated with the absence of forelimb regeneration, this leukopenia is not, of itself, a sufficient condition to inhibit regeneration.
Effect of calcium antagonists on light response of cultured melanophors of Oryzias latipes
Authors: Negishi Sumiko
Published: June 2005

Dispersion of melanosomes in Oryzias melanophores, induced by illumination, was completely inhibited by the Ca2+ antagonists lanthanum (0.2 mM), verapamil (0.3 mM), and papaverine (0.3 mM), but only slightly inhibited by ruthenium red (4 mM). The inhibitory effect of lanthanum or verapamil depended on the extracellular concentration of calcium ions, suggesting the competition between binding of Ca2+ and binding of lanthanum or verapamil. Theophylline induced full dispersion of melanosomes, even in the presence of the Ca2+ antagonists. This fact suggests that Ca2+ is not specifically required for the dispersion of melanosomes per se but, rather, that calcium ions are required for the transduction of light stimulation into cytoplasm. The Ca2+ antagonists affected neither the dark‐induced aggregation of melanosomes nor the aggregation induced by melanophore concentrating hormone.
Differences in the macromolecular composition of the zona pellucida isolated from pig oocytes, eggs, and zygotes
Authors: Hedrick Jerry L. , Wardrip Nathan J. , Berger Trish
Published: June 2005

The macromolecular differences in the zona pellucida (ZP) isolated from pig oocytes, eggs, and zygotes were investigated using two‐dimensional polyacrylamide gel electrophoresis. The ZP was isolated from individual cells or zygotes using micropipettes, radiolabeled with 125I and analyzed using disulfide bond reducing and nonreducing conditions. The reduced ZP isolated from oocytes was composed of four glycoprotein components. The gel pattern of the ZP isolated from a single oocyte was indistinguishable from that isolated en masse. The ovulated egg ZP contained the four oocyte components plus three additional macromolecules. Relative to the egg ZP, the zygote ZP lacked one component but had three additional smaller macromolecules. We concluded that: 1) the macromolecular differences between the oocyte and egg ZPs are caused by the addition of macromolecules to the ZP as the egg transits the oviduct, 2) the macromolecular differences between the egg and the zygote ZPs reflect hydrolytic processing of ZP glycoproteins probably by enzymes derived from the egg cortical granules, and 3) the microheterogeneity of the pig ZP glycoproteins is due to posttranslational modification and is not due to population genetic variation.
Formation of germ layers and roles of the dorsal lip of the blastopore in normally developing embryos of the newt Cynops pyrrhogaster
Authors: Imoh Hiroshi
Published: June 2005

Three‐dimensional relationships between tissues during the formation of germ layers were studied in sections of normally developing embryos of the newt, Cynops pyrrhogaster. In gastrulae, the inner postinvolution layer was not in direct contact with the outer preinvolution layer as a result of the presence of an intervening layer of cells. Only after the formation of the yolk plug, a narrow strip of primitive notochord, which consisted of columnar cells, established a close contact with the central part of the overlaying presumptive neural plate. The primitive notochord was also linked to endoderm at its right and left margins, facing the archenteron. Mesodermal cells other than notochord cells were mesenchymal until the neurula stage, when primitive somites appeared on both sides of the notochord. From a comparison of the relative locations of tissues in embryos at different stages of development, it was shown that the notochord elongates by a remodeling of the mass of the primitive notochord, and that, as the anteriorly directed translocation of the neural area and the invagination of endoderm occur, these processes keep pace with the elongation of the notochord. These observations suggest organizing or guiding roles for the notochord in the formation of germ layers. A role for the dorsal lip of the blastopore as the organizer is discussed in relation to the origin of the notochord.
Seasonal modulation of androgen synthesis in the mink (Mustela vison) is associated with qualitative changes in testicular steroidogenesis in vitro
Authors: Tähkä Klaus M. , Teräväinen Tommi , Sundqvist Christer
Published: June 2005

Annual changes in testicular weight and microsomal androgen synthesis were studied in the mink (Mustela vison). Testicular samples were taken at different phases of the annual reproductive cycle. Steroidogenesis was assessed by investigating seasonal differences in the testicular conversion of [4‐14C]pregnenolone (P) and [4‐14C]dehydroepiandrosterone (DHA) to metabolites in vitro. The steroid metabolites were identified by thin‐layer chromatography. Seasonal changes in the capacity of the testis to produce testosterone from DHA and P correlated positively with the annual reproductive cycle of the species as well as with our earlier findings on plasma testosterone levels. Although the percentage conversion to testosterone (T) from both substrates decreased (39.8% to 19%, DHA; 33% to 15.5%, P) during testicular regression, the in vitro production of androstenedione (A) was increased. The A/T ratio changed from 0.32 (DHA) and 0.42 (P) during testicular activation in November to 1.9 (DHA) and 2.52 (P) during regression in September. Both substrates were utilized efficiently throughout the reproductive cycle. During testicular regression the decreased conversion to T was associated with a marked qualitative and quantitative increase (per unit weight) in the production of unidentified metabolites. The conversion to 5α‐reduced androgens appears to be insignificant, since none of the main 5α‐reduced testicular androgens were identified at any phase of the reproductive cycle.

Our data suggest that the observed differences in testosterone production in vitro are in part due to changes in the activity of 17β‐hydroxysteroid dehydrogenase and possibly also in the enzymes catalyzing the conversion of C21 steroids to androgens. It would also appear that seasonal modulation of androgen synthesis in this species is associated with a prominent qualitative shift in testicular steroidogenesis rather than with a marked reduction in the microsomal capacity to use the substrates.
Stimulation of prostaglandin synthesis in fish follicles by a phorbol ester and calcium ionophore
Authors: Goetz Frederick William , Hsu Sheau‐Yu , Selover Amy
Published: June 2005

The effects of the phorbol ester, phorbol 12‐myristate‐13‐acetate (PMA), and the calcium ionophore, A23187, on prostaglandin E (PGE) and F (PGF) levels were investigated using incubates of brook trout (Salvelinus fontinalis) and yellow perch (Perca flavescens) ovarian tissue. The combination of PMA (0.1 μg/ml) and A23187 (0.1 μg/ml) synergistically stimulated an increase in PGE and PGF levels in incubates of brook trout follicles obtained from females prior to germinal vesicle breakdown, prior to ovulation and postovulation. The highest levels were observed in incubates of follicles taken just prior to ovulation. By itself, PMA also stimulated PGE and PGF accumulation while A23187 was much less effective. A23187, but not PMA, stimulated in vitro ovulation of brook trout follicles. Both cycloheximide and actinomycin inhibited PGE and PGF accumulation in incubates of preovulatory brook trout follicles stimulated with PMA but these inhibitors did not block ovulation induced by A23187 or PMA/A23187.

The effect of PMA and A23187 were tested on the following 3 yellow perch tissue preparations: 1) isolated follicles [free of extrafollicular tissue (EF)], 2) EF tissue, and 3) intact tissue (containing undissected follicles attached to EF tissue). Incubates with all tissue types were performed in the presence or absence of 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐PG, 0.1 μg/ml). PMA (0.1 μg/ml) in combination with A23187 (0.01 μg/ml) stimulated PGE and PGF accumulation in all tissue types though the greatest increase was generally observed in intact preparations. There was a lower accumulation of PGE in intact and EF tissue incubates stimulated with 17,20β‐PG and PMA. In addition, in incubates of ovarian tissue from several perch there was a higher accumulation of PGF in intact preparations stimulated by PMA or PMA/A23187 in the presence of 17,20β‐PG.

The results for both species indicate that phorbol esters (PEs) can stimulate prostaglandin synthesis in follicles and, given that PEs have been shown to stimulate protein kinase C, it is possible that this enzyme is naturally involved in the control of ovarian PG synthesis in fish. It also appears that the stimulation is related to maturational stage in brook trout and steroid stimulation in yellow perch.
Ambystoma maculatum gastrulae have an oriented, fibronectin‐containing extracellular matrix
Authors: Johnson Kurt E. , Darribère Thierry , Boucaut Jean‐Claude
Published: June 2005

During early development of the urodele Ambystoma maculatum, the appearance and distribution of fibronectin‐containing fibrillar extracellular materials were studied by immunocytochemistry. Fibronectin (FN) first appears in the early blastula (stage 7) as thin punctate fibrils on the cell surface concentrated in the marginal zone. In late blastulae (stage 9), thin fibrils are found throughout the blastocoel roof. Early gastrulae (stage 10) have numerous fibrils and multifibrillar strands concentrated in the dorsal lip region and oriented preferentially along a line parallel to the dorsal lip‐animal pole axis. There is a striking increase in the amount of FN fibrils during the rest of gastrulation. This FN‐containing network can be transferred to plastic substrata with preservation of the preferential orientation observed in vivo. Dorsal marginal zone explants placed on such conditioned substrata show polarized outgrowth toward the animal pole region of conditioned areas when placed on the dorsal lip side or the ventral marginal zone side of conditioned substrata. This outgrowth occurs symmetrically on bovine plasma FN‐coated substrata, is prevented by Fab' fragments of antibodies to FN but fails to occur on laminin coated substrata. When migrating mesodermal cells from early gastrulae are cultured on substrata conditioned by deposition of the fibrillar matrix, these cells exhibit striking contact inhibition of locomotion, a phenomenon that may explain dispersal of migrating mesodermal cells across the blastocoel roof. When leading edges of mesodermal cells collide, cells abruptly change direction. When leading edges collide with trailing edges, the trailing edges detach from the substratum and cells move apart in the direction of the leading edge. © 1992 Wiley‐Liss, Inc.
A high molecular weight proteoglycan is differentially expressed during development of the mollusc Concholepas concholepas (Mollusca; Gastropoda; Muricidae)
Authors: Brandan Enrique , González Mauricio , Inestrosa Nibaldo C. , Tremblay Colette , Urrea Rodrigo
Published: June 2005

Incorporation of radioactive sulfate to hatched veliger larvae of the gastropod muricid Concholepas concholepas indicated that over 87% of the sulfated macromolecules were found in the detergent insoluble fraction, rich in extracellular matrix (ECM) components. The sulfated material was solubilized with guanidine salt followed by urea dialysis and fractionated by DEAE‐Sephacel chromatography. Three sulfated compounds eluting at 0.7, 1.1, and 3.0 M NaCl, called peaks I, II, and III, respectively, were obtained. The sulfated compound present in peak I was degraded by pronase or sodium alkaline treatment to a small sulfated resistant material, suggesting the presence of a proteoglycan (PG). Filtration analysis on Sephacryl S‐500 and SDS‐PAGE of the intact PG indicates that it has a high molecular weight (360,000 to over 1 × 106). Monoclonal antibodies (mAb) against this PG were produced. The specificity of one mAb, the 6H2, was demonstrated by size chromatography and ELISA analysis. The epitope recognized by this mAb seems to be present in the core protein of the PG. Both the extent of sulfation and the presence of different sulfated species of PGs were evaluated during the development of this mollusc. A twelvefold increase in the incorporation of sulfate to PGs per milligram of protein was found in veliger larvae compared to blastula‐glastula stages. This change correlated well with the differential expression of the sulfated PG present in peak I. Biochemical and immunological analysis indicate that high levels of this PG are found in veliger and trocophore larvae in comparison with blastula‐gastrula and early juveniles. These results indicate that a high molecular weight PG probably of the ECM is differentially expressed during the development of the gastropod Concholepas concholepas. © 1992 Wiley‐Liss, Inc.
Mechanism of tongue protraction during prey capture in the spadefoot toad Spea multiplicata (Anura: Pelobatidae)
Authors: O'Reilly Susan R. , Nishikawa Kiisa C.
Published: June 2005

Recent studies have used muscle denervation experiments to examine the function of muscles during feeding in frogs. By comparing the results of denervation experiments among taxa, it is possible to identify evolutionary changes in muscle function. The purpose of this study was to examine the function of jaw and tongue muscles during prey capture in Spea multiplicata, a representative of the superorder Mesobatrachia. All members of this group possess a disjunct hyoid apparatus. We predicted that Spea would possess a novel mechanism of tongue protraction on the basis of its hyoid morphology. High‐speed video motion analysis and muscle denervation were used to study the feeding behavior and mechanism of tongue protraction in Spea. Although Spea possesses a relatively long tongue, its feeding behavior is similar to that of short‐tongued frogs of similar body size. Denervation of the m. submentalis had no effect on feeding behavior. When the m. geniohyoideus was denervated, the tongue pad was raised and moved forward slightly, but did not leave the mouth. When the m. genioglossus was denervated, the tongue pad was raised slightly, but no forward movement of the tongue occurred. A similar result was obtained after the mm. genioglossus and geniohyoideus were denerva ed simultaneously. Thus, both the mm. genioglossus and geniohyoideus are necessary for normal tongue protraction in Spea. In contrast, only the m. genioglossus is necessary for normal tongue protraction in archaeobatrachians and neobatrachians. We hypothesize that the disjunct hyoid is responsible for the greater role of hyoid movement during feeding in mesobatrachians. © 1995 Wiley‐Liss, Inc.
Menger's theorem for infinite graphs with ends
Authors: Bruhn Henning , Diestel Reinhard , Stein Maya
Published: June 2005

A well‐known conjecture of Erdős states that given an infinite graph G and sets A, ⊆ V(G), there exists a family of disjoint A − B paths 𝓅 together with an A − B separator X consisting of a choice of one vertex from each path in 𝓅. There is a natural extension of this conjecture in which A, B, and X may contain ends as well as vertices. We prove this extension by reducing it to the vertex version, which was recently proved by Aharoni and Berger. © 2005 Wiley Periodicals, Inc. J Graph Theory 50: 199–211, 2005
Studies on the ketone‐catalyzed decomposition of caroate in aqueous alkaline medium
Authors: Selvararani S. , Medona B. , Ramachandran M. S.
Published: June 2005

The kinetics of ketone‐catalyzed decomposition of caroate (peroxomonosulfate) was studied in aqueous alkaline medium at 25°C. The rate follows simple second‐order kinetics, first order in each [Ketone] and [PMS]. The rate constant values are independent of hydroxide ion concentrations over the range from 0.05 M to 0.15 M. The experimental results suggest that the nucleophilic addition of SO ion at the carbonyl carbon leads to the formation of oxirane intermediate in the rate‐determining step. Oxirane reacts rapidly with another SO to give the parent ketone, oxygen, and SO. The observed substituent effect on the activation energy EA and Arrhenius (A) factor suggests that the electron‐donating substituents stabilize the activated state and make the entropy of activation more negative. This can be explained by the fact that the mechanism of oxirane formation shifts from concentric to nonconcentric process as we go from acetone to 3‐hexanone. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 483–488, 2005
Synthesis of polyetherols with purine ring. Kinetics and mechanisms of reactions, part 2: Reactions in the presence of triethylamine
Authors: Cisek‐Cicirko Irmina , Lubczak Jacek
Published: June 2005

The kinetics and mechanism of reaction between hydroxymethyl derivatives of uric acid and oxiranes in the presence of triethylamine as catalyst was studied. The formation of polyetherols was inhibited in initial steps of the reaction. Two different mechanisms were established for the reactions in which intermediates contained O‐hydroxymethyl and O‐(2‐hydroxyethyl) groups. © 2005 Wiley Periodicals, Inc. Int J Chem Kinet 37: 472–482, 2005