bluuebunny/crossref_metadata_2025 huggingface dataset:
The histogenesis of the rat adrenal cortex: A study based on histologic analysis of mosaic pattern in chimeras
Authors: Iannaccone P. M. , Weinberg W. C.
Published: June 2005

A series of chimeric rats was used to investigate the histogenesis of the adrenal cortex. These animals were produced by amalgamating preimplantation embryos of two congenic strains which express different alloantigens of the major histocompatibility system. The manner in which cells assort themselves during embryogenesis and organ renewal is amenable to analysis by using radiolabeled antibodies directed to the class I antigens. Mosaic pattern analysis of the adrenal cortex of these rats revealed that a clonal pattern of division was maintained across all three histogenic zones of the organ, even in highly unbalanced chimeric combinations. No apparent relationship existed between the proportion of cell types and the area of contiguously similar lineage (patch size). In the series examined, the percentage of cells derived from the PVG‐RT1alineage varied from 5 to 85%. The number of parallel cords varied from 6 to 28. The pattern analysis of informative corners of cross sections of the gland supports the thesis that organ maintenance is the result of cell division from the outside of the cortex toward the inside medullary surface.
Scanning electron microscope studies of sperm incorporation into the zebrafish (Brachydanio) egg
Authors: Wolenski Joseph S. , Hart Nathan H.
Published: June 2005

Morphological studies on the gametes and entry of the spermatozoan into the egg of the zebra danio, Brachydanio rerio, were conducted primarily with scanning electron microscopy. The spermatozoan showed a spherical head, which lacked an acrosome, a midpiece containing several mitochondria, and a flagellum. Observations of the unfertilized egg confirmed and extended prior studies showing a distinct cluster of microvilli on the plasma membrane, identified as the sperm entry site, beneath the inner micropylar aperture (Hart and Donovan, '83). The fertilizing spermatozoan attached to the sperm entry site within 5 seconds of the mixing of a gamete suspension. Binding to the egg microvilli appeared restricted to the equatorial surface of the spermatozoan. Fusion between the plasma membranes of the interacting gametes was followed by the formation of a distinct, nipple‐shaped fertilization cone. The sperm head was partially incorporated into the fertilization cone cytoplasm by 60 seconds postinsemination. The incorporation of the entire sperm head, midpiece, and a portion of the flagellum occurred between 1 and 2 minutes. During this time, the fertilization cone shortened and was transformed into a massive, blister‐like cytoplasmic swelling. Concurrently, upward movements of the ooplasm resulted in the gradual disappearance of the original depression in the egg surface containing the sperm entry site. The second polar body, fully developed by 10 minutes postinsemination, formed approximately 10–15 μm from the site of sperm penetration. Development of the fertilization cone, formation of the second polar body and exocytosis of cortical granules at the sperm entry site readily occurred in parthenogenetically activated eggs, indicating that these surface rearrangements do not require sperm binding and/or fusion.
An ecdysiotropic factor from brains of Heliothis virescens induces testes to produce immunodetectable ecdysteroid in vitro
Authors: Loeb M. J. , Brandt E. P. , Woods C. W. , Borkovec A. B.
Published: June 2005

A factor extracted from brains of male larvae and pupae of the tobacco budworm, Heliothis virescens, caused immature testes to initiate immunoreactive ecdysteroid production in vitro. High–pressure size–exclusion chromatography removed endogenous ecdysteroids from most extract frac–tions. However, active fractions could only be detected when ecdysteroid (as 20‐hydroxyecdysone) was reincorporated into the bioassay medium. The active ecdysiotropin was susceptible to protease degradation. It may be a peptide of approximately 6–12 kD.
Uricoteley: Its nature and origin during the evolution of tetrapod vertebrates
Authors: Campbell James W. , Vorhaben Jean E. , Smith Darwin D.
Published: June 2005

The hepatic mechanism for detoxication of ammonia formed during amino acid gluconeogenesis in uricotelic vertebrates requires the intramitochondrial synthesis of glutamine by glutamine synthetase. This glutamine then serves as a precursor of uric acid in the cytosol. The evolutionary development of uricoteley thus required the localization of glutamine synthetase in liver mitochondria. The mechanism for the mitochondrial import of glutamine synthetase in uricotelic vertebrate liver is not yet known. Tortoises, extant relatives of the stem reptiles, possess both the ureotelic and uricotelic hepatic systems. It therefore seems likely that the genetic events allowing the mitochondrial localization of glutamine synthetase in liver occurred in the amniote amphibian ancestors of the stem reptiles. The selection of ureoteley by the theropsids and of uricoteley by the sauropsids were major events in the divergence and subsequent evolution of these two lines. Once established in the sauropsid line, uricoteley has persisted through to the higher reptiles, crocodilians, and birds. Uricoteley was in part responsible for the radiation of the archosaurs during the Triassic as a water‐conserving mechanism in the adult, thereby allowing them to invade the arid environments of that period. Contrary to dogma, uricoteley was probably of minor significance in the development of the cleidoic egg. Neither mammalian nor avian embryonic liver tissues catabolize amino acids to any great extent, so it is inappropriate to attribute to them a kind of “waste” nitrogen metabolism.
Periodic changes in the organs involved in the erythropoiesis of anemic newts
Authors: Frangioni Giuliano , Borgioli Gianfranco
Published: June 2005

In Triturus cristatus carnifex (Laurenti) newts rendered to tally anemic by treatment with acetylphenylhydrazine (APH) diluted in their tank water (25 mg/liter for 48 hours, with four changes) the recovery of erythron occurs through periodic cycles of mitotic activity in the erythropoietic tissue. These cycles determine a marked increase in blood erythrocyte concentration at regular intervals of about 1 month. The consequence of this trend is the alternation of ferritin and hemosiderin accumulation phases during periods of stasis with iron mobilization phases during periods of erythropoietic activity, which is particularly evident in the Kupffer cells of the liver. Iron mobilization and erythropoietic activity are strictly related to the periodic hypertrophy of some Bowman's capsule cells in the renal corpuscle, which were previously denominated “lactate sensitive cells” (LSC). The histochemistry, location, and behavior of LSC indicate that they are probably the site of erythropoietin production in the newt.
Intergeneric gene transfer of six isozyme loci in rainbow trout by sperm chromosome fragmentation and gynogenesis
Authors: Disney Jane E. , Johnson Kenneth R. , Thorgaard Gary H.
Published: June 2005

Transgenic rainbow trout were produced by fertilizing albino rainbow trout eggs with gamma‐irradiated brook trout (ST) or rainbow trout (RT) sperm and then heat shocking the eggs at 29°C for 10 minutes to induce second polar body retention. Electrophoretic analyses of the RT × ST crosses confirmed the expression of paternal (brook trout specific) alleles at Ldh‐4, Mdh‐3,4, Mdh‐2, Aat‐1,2, Gpi‐3, and 6‐Pgd; the transfer frequency of these loci varied from 2 to 17%. These loci range from 8 to 48 cM from the centromere; proximity of introduced genes to a centromere does not appear to be a factor in retention and expression of foreign genes. Cytogenetic analyses of these same crosses revealed variable numbers and types of chromosome fragments and rings within individuals. Survival and pigment transfer in the resultant gynogenetic offspring varied considerably due to the individual female used in a particular cross. This female effect may have obscured effects due to dose of irradiation and species of male.
Pheromone strains of the Cosmopolitan pest, Nezara viridula (heteroptera: Pentatomidae)
Authors: Aldrich J. R. , Oliver J. E. , Lusby W. R. , Kochansky J. P. , Lockwood J. A.
Published: June 2005

Sexually mature males of the sourthern green stink bug, N. viridula, release a pheromone attractive in the field to conspecific females, males, and late‐stage larvae and to the parasitoid Trichopoda pennipes (Diptera: Tachinidae). The pheromone blend typically includes (Z)‐α‐bisabolene (17%), trans‐ and cis‐1,2‐epoxides of (Z)‐α‐bisabolene (44 and 15%, respectively), (E)‐nerolidol (1.4%), and n‐nonadecane (7.4%). The existence of Nezara pheromone strains, first suggested by differential Trichopoda parasitization of geographically isolated N. viridula populations, has been chemically verified.
Ganglionic circulation and its effects on neurons controlling cardiovascular functions in Aplysia californica
Authors: Furgal Sheryl M. , Brownell Philip H.
Published: June 2005

The abdominal ganglion of the mollusk Aplysia californica receives most of its blood supply through a small caudal artery that branches off the anterior aorta near its junction with the heart. Injection of an ink/gelatin mixture into the caudal artery revealed a consistent pattern of arterial branching within the ganglion and a general proximity of larger vessels to identified neurons controlling circulation in this animal. This morphological arrangement was particularly evident for the heart excitor interneuron, cell L10, which lies next to the caudal artery near its entry into the ganglion. In electrophysiological experiments, L10 was excited when blood flow or oxygen tension within the ganglion was reduced. This effect was expressed as a gradual increase in impulse frequency of L10 and conversion from tonic to bursting mode of spike discharge. L10 follower cells in the RB and LD neuron clusters were affected synaptically by the changes in L10 activity, while other follower cells (L3 and RD neurons) responded independently of L10's synaptic influence. The neurosecretory white cells (R3 to R14) that innervate the major arteries and pericardial tissues were also excited when ganglionic circulation was interrupted. In innervated preparations of the heart and respiratory organs, decreased circulation through the abdominal ganglion stimulated a transient increase in the rate and amplitude of respiratory (gill) pumping and pericardial contractions and caused a sustained increase in activity of the heart. Both responses increase cardiac output and both appear to involve a direct influence of ganglionic circulation on interneurons controlling the gill and heart. These results indicate that the cell‐specific patterns of excitation and inhibition caused by fluctuations in ganglionic circulation may be important factors for maintaining circulatory homeostasis in this animal.
Prolyl 4‐hydroxylase in the foot of the marine mussel Mytilus edulis L.: Purification and characterization
Authors: Marumo Keishi , Waite J. Herbert
Published: June 2005

The mussel foot secretes a variety of unusual hydroxyproline‐containing collagenous and noncollagenous proteins. Prolyl 4‐hydroxylase acting on one or more of the secreted proteins was isolated from the foot by using conventional gel filtration and ion exchange chromatography. Mr of the intact enzyme was 230,000 (α2β2) composed of two subunits with Mr of 60,000 (α) and 57,000 (β) as estimated by HPLC gel filtration and SDS‐PAGE. The enzyme utilized (Pro‐Pro‐Gly)10 as a substrate with an apparent Km value of 0.17 mM. Cofactors and inhibitors were very similar to animal, plant, and microbial prolyl hydroxylases previously described. The enzyme had a relatively sharp pH optimum in the range of 7.8–8.3 and the hydroxyproline formed increased in proportion to the rise in the temperature between 5 and 20°C. No detectable hydroxylation occurred with poly‐L‐proline or the unhydroxylated decapeptide analog (Ala‐Lys‐Pro‐Ser‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys) of the polyphenolic protein. Kinetic studies, however, revealed that the mussel prolyl 4‐hydroxylase was competitively inhibited by poly‐L‐proline and uncompetitively inhibited by the decapeptide. These results suggest that the decapeptide binds the enzymesubstrate i.e. (Pro‐Pro‐Gly)10 complex. It is not yet clear whether this enzyme acts exclusively on collagenous substrates or whether its catalytic purview extends as well to the polyphenolic protein.
Hyperosmotic cellular volume regulation in the ribbed mussel Geukensia demissa: Inhibition by lysosomal and proteinase inhibitors
Authors: Deaton Lewis E.
Published: June 2005

The microbial proteinase inhibitors bestatin, leupeptin, chymostatin, antipain, and pepstatin (all at 10−4 M) inhibit the proteolytic activity of gill homogenates of the ribbed mussel Geukensia demissa by 20–30%. In combination, these inhibitors reduced the hydrolysis of casein by 50%.

Isolated gills from G. demissa acclimated to 500 mOsm seawater (SW) were incubated in 1000 mOsm SW for 8 h in the presence of the following proteinase and lysosome inhibitors: bestatin, pepstatin, leupeptin, antipain, chymostatin, NH4Cl, imidazole, and chloroquine. In control tissues the amino acid pool increased by 155 μmol/g dry wt. Imidazole (10 mM), NH4Cl (10 mM), and chloroquine (10−4 M) inhibited this increase by 70, 60, and 50%, respectively. Pepstatin, bestatin, and leupeptin (all 10−4 M) also inhibited the accumulation of amino acids in gills exposed to higher ambient salinity by 30–40%. In combination, these three microbial proteinase inhibitors reduced the enlargement of the amino acid pool by 60%. Chymostatin and antipain had no effect on the increase in the amino acid content of the tissues.

The effects of these inhibitors on the amino acid levels of gill tissues incubated in isomotic seawater were minor. None of the inhibitors had significant effects on the oxygen uptake of isolated gills.

The results from gills exposed to the amines and chloroquine suggest that lysosome‐mediated proteolysis is involved in the accumulation of alanine and glycine during cellular adjustment to hyperosmotic stress in marine bivalves. The effectiveness of pepstatin, leupeptin, and bestatin in reducing this accumulation implicates aminopeptidase I and cathepsins B and D as the specific enzymes involved in the proteolysis associated with adjustment to hyperosmotic stress.
Studies on the mechanisms of neurulation in the chick: Morphometric analysis of force distribution within the neuroepithelium during neural tube formation
Authors: Nagele Robert G. , Hunter Edward , Bush Kevin , Lee Hsin‐Yi
Published: June 2005

Changes in the shape of neuroepithelial cells, particularly apical constriction, are generally thought to play a major role in generating the driving forces for neural tube formation. Our previous study [Nagele and Lee (1987) J. Exp. Zool., 241:197–205] has shown that, in the developing midbrain region of stage 8+ chick embryos, neuroepithelial cells showing the greatest degree of apical constriction are concentrated at sites of enhanced bending of the neuroepithelium (i.e., the floor and midlateral walls of developing neural tube), suggesting that driving forces resulting from apical constriction are concentrated at these sites during closure of the neural tube. In the present study, we have used morphometric methods to 1) measure regional variations in the degree of apical constriction and apical surface folding at selected regions along the anteroposterior axis of stage 8+ chick embryos, which closely resemble the various ontogenetic phases of neural tube formation, and 2) investigate how forces resulting from apical constriction are distributed within the neuroepithelium during transformation of the neural plate into a neural tube. Results show that, during neural tube formation, driving forces resulting from apical constriction are not distributed uniformly throughout the neuroepithelium but rather are concentrated sequentially at three distinct locations: 1) the floor (during transformation of the neural plate to a V‐shaped neuroepithelium), 2) the midlateral walls (during transformation of the V‐shaped neuroepithelium into a C‐shaped neuroepithelium), and 3) the upper walls (during the transformation of the C‐shaped neuroepithelium into a closed neural tube).
Secondary sex characteristics and connected physiological values in male fallow deer (Dama dama L.) and their relationship to changes of the annual photoperiod: Doubling the frequency
Authors: Schnare H. , Fischer K.
Published: June 2005

Five male fallow deer, who were at the time of casting their first antlers, were maintained in a barn with artificial light control. The daylength sequence applied simulated an amplitude which was normal for 52° latitude, but each cycle lasted only 6 months. This photoperiod produced a doubling in frequency of morphogenetic and connected physiological values so that three complete antler cycles were reached within 19 months. Increase in levels of alkaline phosphatase and in concentrations of total‐cholesterol (TC) and low‐ and high density lipoprotein cholesterol (LDL‐C and HDL‐C) followed the frequency of light alterations and correlated well with antler formation. On the other hand, an increase in neck circumference as well as increase of creatinine levels correlated well with the hard antler period of fertility as well.
Eye regeneration in the mystery snail
Authors: Bever Michele Miller , Borgens Richard B.
Published: June 2005

Mystery snails (Family Ampullariidae) are aquatic prosobranchs which possess structurally complex eyes at the tip of a cephalic eyestalk. No other sensory organs are found in association with this stalk. These snails possess the ability to regenerate the eye completely after amputation through the mid‐eyestalk. Amputation induces gross changes in the cellular character of the entire eyestalk; in particular, an invagination of integumentary epithelium at the apex of the eyestalk stump produces a shallow cleft or “eyecup.” Differentiation of all components of the eye apparently occurs by transdetermination of these epithelial cells. Retinal differentiation and the appearance of a new lens is observed as soon as 14 days postamputation. Complete eyes (by external observation), although smaller than the originals, have regenerated by 25 days postamputation. We compare this regeneration to the reconstruction of other animal tissues, in particular the regeneration of amphibian limbs.
Evidence for the sexual bipotentiality of spermatogonia in the fish, Oryzias latipes
Authors: Shibata Naoki , Hamaguchi Satoshi
Published: June 2005

The origin of the oocyte‐like cells (testis–ova) induced by the administration of estradiol in the testis of the fish, Oryzias latipes, was examined by light and electron microscopy. A small number of testis–ova at the zygotene stage was seen on the sixth day after the beginning of treatment with estradiol, and on the 12th day testis–ova at the pachytene and diplotene stages were observed. Ultrastructural observations revealed that the testis–ova at the zygotene stage were organized as cysts and that all testis–ova within a given cyst developed synchronously. The number of these testis–ova in each cyst varied but cysts containing 16–20 or 26–30 testis–ova were most frequently observed. Spermatogonia of O. latipes are classified morphologically into two types: the type A and the type B Spermatogonia (Grier, '76 Cell Tissue Res., 168:419–431). Type A spermatogonia, which were separated from each other by Sertoli cells, were of the stem type and type B Spermatogonia appeared to divide clonally 9–10 times in each cyst before entering meiosis. The present observations on zygotene testis–ova indicate that the testis–ova originate from type B Spermatogonia which have divided clonally four or five times, suggesting that male germ cells remain sexually bipotent through the early stages of development of type B Spermatogonia.
Induction of maturation in cumulus cell‐enclosed mouse oocytes by follicle‐stimulating hormone and epidermal growth factor: Evidence for a positive stimulus of somatic cell origin
Authors: Downs Stephen M. , Daniel Susan A. J. , Eppig John J.
Published: June 2005

The efficacy of follicle‐stimulating hormone (FSH), epidermal growth factor (EGF), and dibutyryl cGMP (dbcGMP) as inducers of germinal vesicle breakdown (GVBD) in cumulus cell‐enclosed mouse oocytes was examined when meiotic arrest was maintained in vitro with purines, dibutyryl cAMP (dbcAMP), or the phosphodiesterase inhibitor, 3‐isobutyl‐1‐methylxanthine (IBMX). When FSH was added to hypoxanthine (HX)‐containing medium, the effect on oocyte maturation was at first inhibitory and later stimulatory. EGF stimulated GVBD at all time points tested. FSH and EGF also induced GVBD when oocytes were arrested with dbcAMP, IBMX, or guanosine. Dibutyryl cGMP stimulated GVBD when meiotic arrest was maintained with HX, but not when oocytes were meiotically arrested with guanosine, and was inhibitory in dbcAMP‐supplemented medium. FSH and dbcGMP produced a transient delay of oocyte maturation in control medium, but the FSH effect was much more pronounced. EGF had no effect on maturation kinetics. The actions of FSH and EGF required the presence of cumulus cells. Both agents significantly stimulated cAMP production in oocyte‐cumulus cell complexes. A brief exposure of complexes to a high concentration of dbcAMP induced GVBD, suggesting that FSH and EGF may act via a cAMP‐dependent process. The frequency of FSH‐ and EGF‐induced GVBD in cumulus cell‐enclosed oocytes was significantly higher than the frequency of GVBD when oocytes were cultured while denuded of cumulus cells. Thus, the induction of maturation is apparently not mediated solely by oocyte‐cumulus cell uncoupling and termination of the transfer of an inhibitory meiotic signal from cumulus cells to the oocyte. The data suggest the generation of a positive signal within cumulus cells in response to hormone treatment that acts upon the oocyte to stimulate GVBD in the continued presence of inhibitory factors.
Development of golden hamster embryos through the two‐cell block in chemically defined medium
Authors: Schini Scott A. , Bavister Barry D.
Published: June 2005

The effect of increasing the embryo:medium volume ratio on overcoming the hamster two‐cell block was examined. Two‐cell golden hamster embryos from each superovulated female were cultured in microdrops (estimated at 0.75 μ1) or 100 μl macrodrops of chemically defined medium (modified Tyrode's solution [TLP] plus glutamine, isoleucine, methionine, phenylalanine, and taurine). In 11 trials (i.e., with embryos from 11 donors), 28.6% of 269 embryos developed to the four‐cell stage in microdrops, whereas only 2 (0.7%) embryos developed in the macrodrops. When two microdrops were used to culture the two‐cell embryos from each donor (n = 8), 17.8% of 304 embryos developed to four cells. Increasing the embryo:medium volume ratio further by culturing all of the embryos from each donor (n = 10) in single microdrops resulted in 53.1% of 397 embryos developing to four cells. Conditioning of the culture medium by these embryos could not be demonstrated. Increasing the embryo:medium volume ratio may protect against loss of some intracellular component essential for growth of early‐stage hamster embryos. Alternatively, increasing this ratio may permit embryos to reduce the concentration of a substance detrimental to their growth. This work represents the first report of cleavage of hamster two‐cell embryos in vitro. These findings are a significant step towards our goal of obtaining complete preimplantation developmental of hamster embryos in vitro and may be helpful for solving the in vitro developmental blocks in embryos from other species.
Gonadotropin action on androgen synthesis in short‐term incubation of explants and dispersed cells of the testis of Xenopus laevis
Authors: Lecouteux Alain
Published: June 2005

Testis cells of the toad Xenopus laevis were dissociated with collagenase and the cell suspension was enriched for steroidogenic cells by Percoll gradients. Results suggested that cells should be preincubated during a 6‐h period before stimulation with gonadotropin. Our results indicate that a 2‐h incubation period with gonadotropin was necessary to obtain a significant response. Furthermore, the cells can be maintained in a functional state longer than mammalian testis cells. Different gonadotropins were used to stimulate androgen production, and their effects were compared in both dissociated cells and testicular explants. Cells were more sensitive to luteinizing hormone (LH) and follicle stimulating hormone (FSH) than the explants (ED50 LH = 0.041 ± 0.003 μg for cells and 0.097 ± 0.002 μg for explants: ED50 FSH = 0.41 ± 0.03 μg for cells and 0.63 ± 0.03 μg for explants). Moreover, human chorionic gonadotropin (hCG) which only stimulates testicular explants at high doses, failed to stimulate the androgen production of dissociated cells; this indicates a low sensitivity of amphibian testis to hCG and a possible damaging effect of collagenase on the receptors of isolated cells.
Thyrotropic activity of salmon pituitary glycoprotein hormones in the Hawaiian parrotfish thyroid in vitro
Authors: Swanson Penny , Grau E. Gordon , Helms Lisa M. H. , Dickhoff Walton W.
Published: June 2005

The thyrotropic activities of salmon pituitary extract, thyroid‐stimulating hormone (TSH), gonadotropins (GTH), and glycoprotein fractions obtained during purification of salmon TSH and GTH were measured using the parrotfish thyroid culture system. Purified salmon TSH was approximately 1,000 times more potent than bovine TSH in stimulating thyroxine release into the culture medium. Most of the forms of salmon GTH had no thyrotropic activity. One of the forms of salmon GTH (GTH‐F) and three chromatofocusing fractions (CF‐B, ‐C, and ‐E) that were devoid of activity in the coho salmon in vivo had some thyrotropic activity in the parrotfish thyroid culture. Whether the activity of these fractions was due to contamination with TSH, less potent forms of TSH, or inherent thyrotropic activity of a form of GTH is discussed. These results indicate that the parrotfish thyroid culture system can be used to detect thyrotropic activity of fractions obtained during the purification of teleost TSH.
Histochemical and biochemical characterization of two slow fiber types in decapod crustacean muscles
Authors: Mykles Donald L.
Published: June 2005

Myofibrillar proteins in muscles of the claws and abdomen of lobster, Homarus americanus, and the claws of fiddler crab, Uca pugnax, and land crab, Gecarcinus lateralis, have been analyzed with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Fibers contained numerous isoforms of structural and regulatory proteins in assemblages correlated with fiber type. One fast (F) and two slow (S1 and S2) fibers were identified. All F fibers possessed two isoforms of paramyosin (P1 and P2), while all slow fibers, with the exception of Uca major claw, contained only the P2 variant. S1 and S2 fibers were distinguished by the distribution of a large isoform of troponin‐T (T1; Mr = 55,000); S2 fibers in all three species contained T1 in addition to one or two smaller‐molecular‐weight variants usually associated with S1 fibers. In order to determine whether the slow fibers differed in histochemical properties, land crab claw closer muscle was cryosectioned and stained for myofibrillar ATPase and NADH diaphorase activities. Most S2 fibers had lower ATPase and higher NADH diaphorase activities than S1 fibers, which indicated that S2 fibers had a lower rate of contraction and were more fatigue‐resistant than S1 fibers. It is proposed that the S1 and S2 fibers defined by biochemical and histochemical criteria are identical to the slow‐twitch and tonic fibers, respectively, characterized physiologically.
Improved growth and development of presomite mouse embryos in whole embryo culture
Authors: Hunter E. S. , Balkan W. , Sadler T. W.
Published: June 2005

A rotator whole embryo culture system was used to assess the growth and development of late‐primitive‐streak‐stage (Theiler stage 9‐10) mouse embryos to the limb‐bud stage of organogenesis in a variety of media containing combinations of mouse serum (MS), rat serum (RS), and Tyrode's buffer (TB). The results demonstrate that embryonic growth and morphogenesis to the early limb‐bud stage (20 somite pairs; 48‐h total culture period) mimicked that in vivo when embryos were grown for 24 h in combinations of MS:RS:TB 1:2:1 or 2:1:1 (v/v/v) and then were transferred to fresh medium containing RS:TB 3:1 at the early somite stage. When the culture period was extended for an additional 24 h (total 72‐h culture period) embryonic growth retardation was observed. Regardless of the medium employed, superior growth was observed in embryos transferred at the early somite stage when compared to embryos cultured continuously in the same medium for the entire 48‐ or 72‐h culture period.
On the diversity of sperm basic proteins in the vertebrates: VI. Cytochemical and biochemical analysis in birds
Authors: Chiva M. , Kasinsky H. E. , Mann M. , Subirana J. A.
Published: June 2005

By cytochemical criteria, the sperm basic proteins in birds are remarkably constant. Type 1 protamines are found in five species of birds representing four orders: Coturnix japonica (quail) and Gallus domesticus (rooster), order Galliformes; Anas platyrhynchos (duck), order Anseri‐formes; Melopsittacus undulatus (budgerigar), order Psittaciformes; Molothrus ater (cowbird), order Passeriformes. This constancy is confirmed by the isolation of electrophoretically typical protamines from sperm nuclei of quail, duck and budgerigar as well as from Carduelis carduelis (goldfinch), another passeriform bird, and Columba livia (pigeon), order Columbiformes. In each species only typical protamines are found in ripe spermatozoa; no histones are present. In quail and pigeon, several protamines of similar molecular weight can be found, whereas in duck, goldfinch, and budgerigar a single protamine component is apparent. An additional basic protein is found in budgerigar, which does not appear to be part of the ripe sperm nucleus. Such additional basic proteins have not been detected in the other species studied. No significant amounts of intermediate proteins that could be involved in spermatid maturation have been detected either cytochemically or biochemically in any of the species studied. The overall similarity amongst protamines from seven birds representing five orders might be related to the uniform mode of reproduction within the class Aves, consisting of internal fertilization and egg laying.
Induction of spawning and oocyte maturation by 5‐hydroxytryptamine in the surf clam
Authors: Hirai Setsuro , Kishimoto Takeo , Kadam A. L. , Kanatani Haruo , Koide S. S.
Published: June 2005

Gamete shedding can be induced in male and female surf clams (Spisula solidissima and sachalinensis) by injecting 5‐hydroxytryptamine (5‐HT) into the gonads. The administration of 0.4 ml of 2.0 mM 5‐HT induced spawning in all the clams tested. Shedding of gametes occurred within 2 to 3 min after the treatment and continued for about 40 min. The majority of the shed oocytes retained their germinal vesicles and underwent maturation upon insemination. The fertilized oocytes developed normally to the trocophora stage. The motility of the shed sperm was normal. 5‐HT added to the suspending medium to the final concentrations of 10 to 100, μM induced maturation of isolated Spisula oocytes in vitro. The oocytes underwent germinal vesicle breakdown, chromosomal condensation, extrusion of the first and second polar bodies, and pronuclei formation. The neurotrans‐mitter‐induced production of maturation‐promoting factor. In conclusion, 5‐HT induces gamete spawning and initiates oocyte maturation in the surf clam.
Chromatin topology during the transformation of the mouse sperm nucleus into pronucleus in vivo
Authors: Garagna Silvia , Redi Carlo Alberto
Published: June 2005

Time relationships of sperm chromatin dispersion and sperm nucleoprotein replacement have been studied in vivo, by an in situ cytochemical approach. We used the Peulgen reaction to reveal DNA, which allows us to record both processes simultaneously, on the basis of the return after fertilization to haploid Feulgen values after sperm nucleoprotein replacement with somatic histones. We have shown that sperm nucleoprotein replacement occurs at around anaphase II, whereas sperm chromatin dispersion is massive between the anaphase and telophase II oocyte phases. The morphological pattern of sperm chromatin dispersion supports the idea that the process involves the whole sperm chromatin mass simultaneously, with the region located between the implantation fossa and the postacrosomial region the last to swell.
Effects of cytochalasins B and D on the fertilization of zebrafish (Brachydanio) eggs
Authors: Wolenski Joseph S. , Hart Nathan H.
Published: June 2005

The effects of selected concentrations of cytochalasins B (1–10 μg/ml; CB) and D (10, 50 μg/ml; CD) on the morphology and fertilization of zebra danio (Brachydanio) eggs were studied primarily with light and scanning electron microscopy. Eggs pretreated with either CB (10 μg/ml) or CD (10, 50 μg/ml) prepared in Fish Ringer's solution‐0.5% DMSO showed a flattened shape, alterations in the form of surface microplicae and microvilli, and occasional spontaneous exocytosis of cortical granules. All eggs preincubated in either CB or CD were activated upon transfer to tap water, showing cortical granule exocytosis, elevation of the chorion, and formation of a fertilization cone. When eggs were pretreated for 5 minutes with 1–5 μg/ml CB or 10 μg/ml CD and inseminated, they incorporated the fertilizing sperm and typically developed to the two‐cell stage. A single sperm cell attached to and fused with the sperm entry site microvilli but failed to enter the cytoplasm in eggs preincubated with 10 μg/ml CB. Eggs that were immersed continuously in either CB (10 μg/ml) or CD (50 μg/ml) 15 seconds after insemination also failed to incorporate the fertilizing sperm. Treatment of eggs after insemination with CD (10 μg/ml), however, did not prevent sperm cell incorporation or fertilization cone formation. Our drug data suggest the presence of actin‐containing filaments in the danio egg before and following fertilization. These filaments appear to play a role in maintaining the shape of the egg cell and its surface specializations and in the incorporation of the fertilizing sperm. The fertilization cone appears to form independently of actin polymerization.
Metabolic fates of lactate during recovery from activity in an anuran amphibian, Bufo americanus
Authors: Withers Philip C. , Lea Malcolm , Solberg Thomas C. , Baustian Mark , Hedrick Michael
Published: June 2005

The aerobic metabolism of Bufo americanus is 0.56 ± S.E. 0.12 ml O2 g−1 h−1; lactate accumulates to 0.63 ± S.E. 0.06 mg lactate g−1, during a 10‐min bout of enforced activity. About 20% of the energy expended during activity is derived from anaerobic metabolism and 80% from aerobic metabolism. Aerobic metabolic rate decreases rapidly after the cessation of activity and reaches resting levels by 1 hr post‐activity. Whole body lactate content declines more slowly and in a linear fashion, reaching resting levels about 1 hr post‐activity. About 7% or less of the 14C‐lactate administered after activity was estimated to have been eliminated by aerobic metabolism of the lactate; the remaining 14C activity was recovered in body tissues and urine or was excreted across the skin. Most tissues examined (liver, skin, ventricle, lungs, stomach, intestine) had similar 14C activities per gram of tissue, but skeletal muscle had a higher 14C activity per gram. Considerable fractions of the tissue 14C content were isolated as glycogen (22–39%), lower fractions as protein (1–24%), and little as lipid (2–4%). There are some differences in the distribution of 14C label, depending on whether 14C‐lactate or 14C‐glucose were administered at rest, or after activity, but little of the 14C label was aerobically metabolised for either substrate. The observed fraction of lactate oxidised by aerobic metabolism (<10% of total 14C lactate) is less than the theoretical value for the most energetically economical metabolism of lactate (17%: a Meyerhof quotient of 5) and considerably less than the fraction of lactate oxidised by mammals (20–90%).
Metabolic responses to cold acclimation in the swimming musculature of lake whitefish, Coregonus clupeaformis
Authors: Blier Pierre , Guderley Helga
Published: June 2005

To evaluate the acclimatory capacity of juvenile lake whitefish (Coregonus clupeaformis), a species which is extensively distributed in northern latitudes and which experiences a fairly wide range of temperatures, we assessed the effect of acclimation at 5 and 18°C on the glycolytic and aerobic capacity of swimming muscle. Cold acclimation during six months does not increase the proportion of red muscle in the swimming musculature or the levels of cytochrome oxidase and citrate synthase, indicating a lack of thermal compensation of the aerobic capacity of the swimming muscles. By contrast, the glycolytic potential of the swimming musculature, as assessed by the activities of hexokinase and phosphofructokinase, displayed positive thermal compensation. This may facilitate the conservation of locomotory capacity at low temperatures.
Surface behavior in artificially constricted sand dollar eggs and egg fragments
Authors: Rappaport R.
Published: June 2005

In mathematical and computer models of cytokinesis in animal cells, it is sometimes assumed that the nature of the surface is such that it will develop furrowing behavior independently of the mitotic apparatus in response to folding and distortion. Similarly, it has been postulated that after the dividing cell has achieved a moderate degree of constriction, the process is completed with little or no additional energy or preorganized structure. The purpose of these experiments was to test those assumptions by artificially constricting the surface of Echinarachnius parma eggs with a pair of hooks at the time of cytokinesis.

When the surface of unnucleated egg fragments or those containing single asters were constricted to 27–30 μm in diameter, no furrowing occurred in the constriction plane, and the fragments immediately rounded up when the hooks were removed. When fragments containing a single aster were reshaped into cylinders, the surface developed astral constrictions. When intact cells were constricted so that the constriction plane did not coincide with the plane of an astral center, no furrowing occurred. When the constriction plane and that of the astral center coincided or when they were very close, furrowing activity developed and then regressed.

These results indicate that the sand dollar egg surface does not tend to develop furrowing activity in response to imposed folds and distortion independently of the mitotic apparatus. They also suggest that single asters can elicit contractile activity in nearby surface.
In vitro fusion of newt macrophages
Authors: Płytycz Barbara , Potter Sandra W. , Cohen Nicholas , Bayne Christopher J.
Published: June 2005

Spontaneous formation of multinucleate giant cells is often observed in in vitro cultures of peritoneal adherent macrophages from the newts, Notophthalmus viridescens and Taricha granulosa (urodele amphibians). The frequency of such giant cells in these cultures is increased by the addition of phorbol myristic acetate at the initiation of the cultures. This high frequency of multinucleate cells permitted us to evaluate whether multinucleate giant cells arise by cell fusion and/or by repeated nuclear division without cytokinesis. Cell fusion is readily detectable by scanning electron microscopy. To determine whether nuclear division without cytokinesis also occurs, some cultures were treated with colchicine to arrest mitotic figures; others were pulsed with tritiated thymidine to detect DNA synthesis. Mitotic figures were not seen in acridine orange‐stained samples. In monolayers that were processed for autoradiography, only a few nuclei were marked with tritium. These observations suggest that nuclear division does not contribute significantly, if at all, to the formation of multinucleate giant cells from cultured newt peritoneal macrophages.
Appearance and regulation of an antigen associated with limb regeneration in Notophthalmus viridescens
Authors: Gordon Herman , Brockes Jeremy P. , Wilson Frank
Published: June 2005

Previous reports from this laboratory have documented a cellular antigen, 22/18, associated with regeneration of forelimbs in the newt Notophthalmus viridescens (Kintner and Brockes: Nature 308:67–69, 1984; Journal of Embryology and Experimental Morphology 89:37–55, 1985; Brockes: Science 225:1280–1287, 1984; Kintner et al.: Molecular Basis of Neural Development, 1985). The antigen is expressed at various stages of regeneration in several types of cells including Schwann cells and muscle cells. We report here that, in addition to the previous results, the expression of 22/18 occurs very early in the regeneration process and is initially confined to an apparently nonmuscle, non‐neural population of cells. The well‐defined context of the antigen's first appearance has allowed us to investigate the regulation of its expression. In particular, injury is sufficient to elicit the appearance of 22/18. It can appear prior to mitosis and despite denervation. In circumstances where regeneration is inhibited, expression of 22/18 can persist.
Localized heat‐shock induction in Drosophila melanogaster
Authors: Monsma Scott A. , Ard Robert , Lis John T. , Wolfner Mariana F.
Published: June 2005

We describe a technique for inducing localized expression of genes fused to heat‐shock gene promoters. We demonstrate that a localized heat‐shock response can be induced in Drosophila melanogaster at any developmental stage after formation of the cellular blastoderm by contacting a region of the animal with a heated needle. The size of the induced region can be altered by varying parameters such as the temperature and size of the needle tip. The test system utilized here is a D. melanogaster strain transformed with a fusion of the Drosophila hsp26 gene and the E. coli lacZ gene; the activity of this hybrid gene is monitored in whole animals by staining for β‐galactosidase activity. Induced β‐galactosidase activity is confined to the cells in the region of heating; the β‐galactosidase activity can still be detected 48 hr after the heat shock.

Given the heat inducibility of Drosophila heat‐shock promoters in heterologous systems, we suggest that this technique will be useful for allowing spatially controlled induction of a gene of interest in any organism into which fusion genes can be introduced. Additional uses of the technique for following cell movements during development are discussed.
Diel metabolic and activity patterns of the lemon shark (Negaprion brevirostris)
Authors: Nixon Asa J. , Gruber Samuel H.
Published: June 2005

Computation of the energy budget, average daily metabolic rate, and standard metabolism require information on hourly metabolic rates over a full 24‐hour period. This study provides such information for the lemon shark, a tropical predator. Simultaneous measurements of metabolism and activity in an annular respirometer demonstrated that oxygen consumption increased from a daytime level of 176 to 210 mgO2 kg−1h−1 at night. Activity increased concurrently from 0.17 to 0.33 bl s−1. The average daily metabolic rate was 16.2 k cal kg−1 at 0.26 bl s−1 and 23°C. Both activity and metabolic rate appear to have endogenous circadian components.
Effect of hyperoxia acclimation on catalase and glutathione peroxidase activities and in vivo peroxidation products in various tissues of the frog Rana ridibunda perezi
Authors: López‐Torres M. , Gil P. , de Quiroga G. Barja
Published: June 2005

Among vertebrates, adult amphibians are known to be especially tolerant to exposure to high environmental oxygen tensions. To clarify the basis for this high O2 tolerance, adult Rana ridibunda perezi frogs were acclimated for 15 days to water‐air phases with either 149 mm Hg O2 (normoxia) or 710 mm Hg O2 (hyperoxia). At the end of the acclimation, various morphometric and biochemical paramaters related to oxidative stress were measured in seven organs and tissues. Hyperoxia acclimation did not change either the total weight of the animals or the total and relative wet weights of the organs studied, except for the brain, which showed weight increases in the hyperoxic group. In vivo tissue peroxidation increased in the kidney; decreased in the skeletal muscle and skin; and did not change in the liver, lung, brain, and heart after hyperoxic exposures. Whereas liver, lung, and skin showed glutathione peroxidase (GSH‐Px) activities with both cumene hydroperoxide (cumene‐OOH) and H2O2 as substrates, skeletal muscle only showed H2O2 GSH‐Px activity. Hyperoxia acclimation did not change either catalase (CAT) or GSH‐Px activities in any organ, except for the liver in which CAT activity was induced by hyperoxia. Thus hyperoxia tolerance in this species does not need the induction of H2O2‐detoxifying enzymes in the majority of the organs. It is suggested that the high O2 tolerance of this amphibian species is related to its comparatively high constitutive GSH‐Px activities.
Development of β‐amino acid transport in the kidney
Authors: Chesney Russell W. , Zelikovic Israel , Dabbagh Shermaine , Friedman Aaron , Lippincott Shirley
Published: June 2005

This study focuses on the maturation of the renal β‐amino acid transport system and uses dietary manipulation as a probe. The epithelial surface of the renal proximal tubule is responsible for the conservation of ions and organic solutes including β‐amino acids. This β‐amino acid transport system is stimulated during periods of reduced dietary intake and permits increased excretion following dietary excess. We have examined transport of the sulfur‐containing β‐amino acid, taurine, as a measure of this renal adaptive response to fluctuations in dietary sulfur amino acid intake and as a substrate for the β‐amino acid transport system.

A precession of taurine uptake values by brush border membrane vesicles (BBMV) prepared from nursing rats from youngest to oldest was evidente. However, these membranes demonstrate the full renal adaptive response to altered sulfur amino acid intake after the first week of life. This adaptive response is expressed at the brush border surface by transport changes in both directions (“up regulation” and “down regulation”), through changes in the initial rate (15 sec) of Na+‐taurine cotransport. No alterations in the lipid microenvironment of the membrane, as detected by altered membrane fluidity, were uncovered. Although vesicles from 7‐day‐old pups demonstrate adaptation and accumulate taurine to a limited extent, the accumulation of Na+, which energizes uptake, may be altered, thereby preventing full expression of the adaptive response and of transport capacity at this age.