bluuebunny/crossref_metadata_2025 huggingface dataset:
Gonadal histology and plasma sex steroids during sex differentiation in grey mullet, Mugil cephalus
Authors: Chang Ching‐Fong , Lan Shih‐Chieh , Chou Hsin‐Yiu
Published: June 2005

Gonadal histology and plasma levels of sex steroids were investigated during the period of sex differentiation and the annual cycle of grey mullet, Mugil cephalus. Primordial germ cells were observed in the gonads of 3‐month‐old grey mullet and appeared to be undifferentiated in fish at 3 to 6 months of age. At 7 to 14 months of age 23.3% of the fish examined had differentiated into males and females whereas 70–90% of 15‐ to 17‐month‐old fish and more than 95% of 18‐ to 24‐month‐old fish had completed gonadal differentiation. Differentiation into females occurred earlier than males. In 15‐ to 17‐month‐old fish, spermatogonia and primary oocytes in the perinucleolus stage were observed in the testes and ovaries, respectively. Testes underwent intensive spermatogenesis at 18 to 24 months whereas ovaries during this period contained both primary and well developed vitellogenic oocytes. There was no sexual dimorphism in the annual cycle of plasma estradiol‐17β (E2), testosterone (T) and 17α‐hydroxyprogesterone (17α‐OH P) in 7‐to 24‐month‐old grey mullet. Plasma E2 levels significantly decreased in 9‐ to 12‐month‐old presumptively female and male grey mullet. On the contrary, peak levels of plasma T were observed in both female and male grey mullet at 10 to 11 months. Plasma 17α‐OH P levels did not show significant profiles in the annual cycle of grey mullet. The data conclude that few gonads at 7 to 14 months begin to differentiate, but sex differentiation mainly occurs at the age of 15 months. It is 9 to 12 months of age when the decreases of plasma E2 and increases of T levels significantly occur in both female and male grey mullet, M. cephalus. © 1995 Wiley‐Liss, Inc.
Effects of acclimation and assay temperature on outer‐ and inner‐ring thyroxine and 3,5,3′‐triiodo‐L‐thyronine deiodination by liver microsomes of rainbow trout, Oncorhynchus mykiss
Authors: Johnston C. E. , Eales J. G.
Published: June 2005

We studied effects of temperature on the deiodinating enzymatic pathways governing hepatic production of 3,5,3′‐triiodo‐L‐thyronine (T3) from L‐thyroxine (T4) in rainbow trout. Kinetics of outer‐ring deiodination (ORD) and inner‐ring deiodination (IRD) of T4 and T3 were measured in vitro at 4°, 12°, and 18°C using liver microsomes of trout that were acclimated for 21 days at 4°, 13°, and 18°C and fed on temperature‐compensated food rations. For all temperatures the Vmax (level of functional enzyme) and Vmax/Km (index of enzyme efficiency) values for the T3‐generating T4ORD pathway greatly exceeded values for the T3‐degrading T3IRD pathway; T4IRD and T3ORD pathways were negligible. The main effects of temperature occurred between 4°C and 12°/13°C; T4ORD Vmax/Km rose from 0.48 at 4°C to 1.62 at 12°/13°C and to 1.83 at 18°C. This response correlated with in vivo deiodination rates measured previously. T4ORD apparent Km did not change over the temperature acclimation range, suggesting an absence of temperature‐induced isozymes. However, Km was depressed at the assay temperature of 4°C, indicating a direct effect of temperature to enhance enzyme‐T4 substrate affinity. T4ORD Vmax was insensitive to assay temperature but was depressed in trout acclimated at 4°C. We conclude that from 4° to 18°C T4ORD is the main thyroid hormone deiodinating pathway in trout liver. T4ORD activity increases more than threefold from 4° to 13°C. This is due to a direct effect of temperature to decrease Km and to an acclimatory increase in Vmax. © 1995 Wiley‐Liss, Inc.
Seasonal fiber growth cycles of ferrets (Mustela putorius furo) and long‐term effects of melatonin treatment
Authors: Nixon A. J. , Ashby M. G. , Saywell D. P. , Pearson A. J.
Published: June 2005

Pelage cycles of ferrets are poorly documented, although it is clear that their timing is sensitive to daylength, mediated by pineal melatonin. Hair follicles were monitored histologically in ferrets from 3 to 19 months of age in order to describe naturally occurring changes in follicle growth status and follicle number over three successive cycles of fur growth. Melatonin was administered to some of these animals in late summer to determine the long‐term effects of perturbation of hormonal control. Circulating melatonin was elevated for approximately 50 days by 8‐mg continuous release implants. Treated animals grew both their first winter coat, and subsequent summer coats 18 days in advance of untreated controls, but this effect did not extend to the second winter coat. Reimplantation the following year induced an advancement of the autumn follicle growth as in the 1st year. Autumn fiber growth occurred at similar times in untreated males and females, and response to melatonin did not differ between sexes. Hair follicle regression and shedding during the natural spring molt was also contemporaneous in males and females, but fiber regrowth occurred 4–6 weeks later in males as compared with females, suggesting that reproduction‐related factors affect fiber growth initiation, and that fiber growth and shedding are physiologically distinct processes. Melatonin implants in autumn also affected reproduction in the spring, advancing oestrus by 3–4 weeks. These results show that interference with photoperiodic and hormonal control mechanisms in ferrets can affect pelage and reproductive cycles for up to 10 months. © 1995 Wiley‐Liss, Inc.
Vitelline coat lysins from molluscan sperm and their use in microinjection
Authors: Solano‐Estrada Lourdes Aidée , Plenge‐Tellechea Luis Fernando , Stephano José Luis , Gould Meredith C.
Published: June 2005

Microinjection through the tough vitelline coats of oocytes from three molluscan species (Haliotis rufescens, Norrisia norrisi, and Astrea undosa) was facilitated by the local application of sperm vitelline coat lysin with a micropipet. Extracts containing lysin were the 12,000g supernates of frozen‐thawed sperm. In H. rufescens and N. norrisi, lysin extracts dissolved a small hole through the vitelline coat, whereas in A. undosa, the lysin created a pathway for microinjection by softening the vitelline coat without dissolving it. Similar effects were observed when oocytes were suspended in lysin extracts: Vitelline coats dissolved completely in H. rufescens (previously reported by Lewis et al. [1982], Dev. Biol. 92:227–239) and N. norrisi, but only softened and swelled in A. undosa (although they could then be removed by mechanical agitation). The lysin extracts were highly species specific and had no visible effects on heterologous vitelline coats even at concentrations higher than those required to dissolve/soften homologous coats. Evidence that N. norrisi lysin acts by a non‐enzymatic mechanism was provided by the observation that mobilities of radioactive bands in sodium dodecyl sulfate polyacrylamide gels were unchanged following dissolution of 125I‐labeled vitelline coats. Electron micrographs of the previously undescribed sperm from N. norrisi and A. undosa are also presented. © 1995 Wiley‐Liss, Inc.
Kinematics of ducklings swimming in formation: Consequences of position
Authors: Fish Frank E.
Published: June 2005

The kinematics of the paddling stroke of ducklings swimming in formation were analyzed to detect differences in relation to swimming effort and position in the formation. Paddling motions of the feet were filmed as ducklings swam in a constant 0.3 m/s water current behind a decoy which could be in the water or suspended above the water. Ducklings were tested in clutches of one, two, and four ducklings at ages of 3, 7, and 14 days of age. Ducklings swam in organized formations with the lead duckling 0.25 body length posterior of the decoy and with an average inter‐duckling distance of 0.1 body length. Stroke frequency was constant within any age and decreased with age. Age, decoy position, and clutch size affected the length of the arc traversed by the foot through the power stroke for the most posterior duckling in the formation. Trailing ducklings had an arc length 16.9% less than leading ducklings, and ducklings swimming in formation had an arc length as much as 29.8% less than a solitary duckling. These results suggest that mechanical energy is conserved when formation swimming is employed. © 1995 Wiley‐Liss, Inc.
Effects of space flight on Xenopus laevis larval development
Authors: Snetkova E. , Chelnaya N. , Serova L. , Saveliev S. , Cherdanzova E. , Pronych S. , Wassersug R.
Published: June 2005

Fifty‐three fertilized Xenopus laevis embryos at early tail bud stage were launched into orbit aboard a Biocosmos satellite and remained in microgravity for 11.5 days. During this period, the embryos hatched and continued to develop as free‐living larvae. Forty‐eight individuals survived the mission. Upon recovery these tadpoles had smaller heads/bodies and proportionately longer tails than ground controls. Almost all the flight animals had caudal lordosis and consequently swam in backward somersaults. Compared to ground‐based controls, their notochords were significantly larger in cross‐sectional area and were deformed. Caudal muscle fibers were less dense and involuted in a fashion indicative of degeneration. In contrast, cranial muscles associated with buccal pumping did not differ between the flight and control animals.

Upon landing, the flight larvae were found to be negatively buoyant and lay on the bottom when they were not swimming. They had significantly smaller lungs than controls, suggesting that they had failed to inflate their lungs in microgravity. Additionally, the branchial baskets, gill filters and thymuses all showed signs of retarded development or degeneration.

The caudal deformity that we observed in the flight X. laevis has been independently observed in three other space flight experiments where embryos were launched then hatched in space. In contrast, Xenopus larvae from another orbital experiment that were raised from fertilization through hatching in space did not exhibit any caudal abnormalities. These divergent results suggest that either features of the launch itself (i.e., high acceleration and vibration) or an abrupt decrease in gravity during the tail bud stage detrimentally affects musculoskeletal development in anurans. © 1995 Wiley‐Liss, Inc.
Intratesticular retention of sperm and premature decline in fertility in the domestic rooster, Gallus domesticus
Authors: Muncher Yossi , Sod‐Moriah Uriel A. , Weil Simy , Rosenstrauch Avi , Friedländer Michael
Published: June 2005

In domestic roosters, which were originally seasonal breeders and that are now kept under unnatural and unchanging conditions throughout the year, fertility peaks at 32 weeks of age (96%) but it subsequently declines rapidly to only 5% at 110 weeks despite the fact that roosters can live for about 10 years. Roosters exhibiting this low‐fertility syndrome have reduced levels of spermatozoa in the ejaculate. Concomitantly, however, superabundant but apparently normal spermatozoa are found attached to Sertoli cells and, in addition, the seminiferous epithelium fails to show evidence of the regression or atrophy that characterizes both aging non‐seasonal breeders and true seasonal breeders during non‐reproductive periods. This syndrome of premature low fertility appears to stem from impaired spermiation with resultant retention of spermatozoa by Sertoli cells. To examine this problem, we compared intratesticular incorporation of 3H‐thymidine between high‐fertility (32‐week‐old) and low‐fertility (82‐week‐old) roosters. Radioactivity associated with spermatozoa, 33 days post‐injection, was almost 50% higher in the low‐fertility roosters than in the high‐fertility ones. By contrast, both groups showed similar characteristics with respect to a) intratesticular incorporation of 3H‐thymidine, b) dynamics of spermatogenesis, c) intratesticular level of radioactivity just before the initiation of spermiation, and d) the duration of both spermatogenesis and the time required for sperm to pass through the genital tract. Our results confirm that intratesticular retention of sperm occurs in roosters with premature low‐fertility syndrome and suggest new possibilities for the study of the complex relationship between Sertoli cells and spermatozoa and the effects of this relationship on fertility. © 1995 Wiley‐Liss, Inc.
Isolation and characterization of opioid peptides in the avian brain
Authors: Kotegawa Takeru , Takahashi Toshio , Tsutsui Kazuyoshi , Ikeda Tetsuya , Minakata Hiroyuki , Nomoto Kyosuke
Published: June 2005

Neuropeptides are supposed to be implicated in the regulation of hormone as well as nonhormone dependent behavioral processes in birds. Previous immunohistochemical studies have suggested that in birds opioid pentapeptides, Met‐ and Leu‐enkephalins, may be present in the brain including the regions that regulate sex hormone dependent behaviors, such as reproductive behaviors. To determine biochemically the presence of opioid peptides in the avian brain, a study was conducted to isolate these peptides from Japanese quails and zebra finches. Acetic acid extracts of the quail and finch brains were respectively forced through disposable C‐18 reversed‐phase cartridges, and then the retained material was subjected to the cation‐exchange and reversed‐phase high performance liquid chromatographic (HPLC) purifications. All of the purified substances showed a single peak on the reversed‐phase HPLC and these substances enhanced spontaneous contractions of the avian rectum. The purified bioactive substances were further subjected to amino acid sequence analysis and were characterized as peptides with the following sequences: Tyr‐Gly‐Gly‐Phe‐Met, Tyr‐Gly‐Gly‐Phe‐Leu, and Tyr‐Gly‐Gly‐Phe‐Met‐Arg‐Phe. These three peptides were identical with opioid pentapeptides, Met‐ and Leu‐enkephalins, and a heptapeptide, Met‐enkephalin‐Arg6‐Phe7, which had been previously isolated from mammalian species. This is the first direct demonstration of the presence of opioid peptides in the avian brain and confirms previous immunohistochemical findings suggesting a functional role for the opioid peptide in neural mechanisms of avian reproductive behavior. © 1995 Wiley‐Liss, Inc.
Effects of opioid peptides on the electrical activity of preoptic and hypothalamic neurons in the quail brain
Authors: Furukawa Yasuo , Kotegawa Takeru , Tsutsui Kazuyoshi
Published: June 2005

We have recently isolated three opioid peptides, i.e., Met‐ and Leu‐enkephalins and Met‐enkephalin‐Arg6‐Phe7, from the avian brain. In the present study, therefore, effects of these endogenous opioid peptides on the electrical activity of preoptic and hypothalamic neurons of the adult male Japanese quail were examined using a brain slice preparation. All of the three opioid peptides inhibited the spontaneous firing activities of subsets of neurons in the preoptic area and the paraventricular nucleus in the hypothalamus. Threshold concentrations for the inhibitory action were between 10−7 and 10−6 M in Met‐ and Leu‐enkephalins and approximately 10−6 M in Met‐enkephalin‐Arg6 ‐Phe7, respectively. In a few cells in these brain areas, however, Leu‐enkephalin rather potentiated the spontaneous activities, resulting in an increase of firing rates or a decrease of interburst intervals. The inhibitory effect of Met‐enkephalin was completely blocked by naloxone, an opioid receptor antagonist, but not affected by bicuculline, a γ‐aminobutyric acid A (GABAA) receptor antagonist. These results suggest that there are functional opiate receptors in subsets of preoptic/hypothalamic neurons and that one of their main physiological functions in these areas is an inhibition of neuronal activities. Because these brain regions are considered to be involved with the regulation of a variety of male reproductive behaviors, opioid peptides may regulate some reproductive behavior through the mechanism that provokes such an inhibition. © 1995 Wiley‐Liss, Inc.
Evidence for intracellular stores of calcium ions involved in regulating nematocyst discharge
Authors: Russell Thomas J. , Watson Glen M.
Published: June 2005

In sea anemones, nematocyst discharge is regulated in part by chemical substances derived from prey. Adding N‐acetylated sugars or proline to seawater sensitizes cnidocytes to discharge nematocysts. Extracellular calcium ions are required since discharge is inhibited by reducing the Ca2+ concentration in artificial seawater. Known inhibitors of L‐type Ca2+ channels, nifedipine and verapamil, reduce discharge sensitized by N‐acetylated sugars but not by proline. Conversely, known inhibitors of certain Ca2+ channels at intracellular storage sites, ryanodine and procaine, reduce discharge sensitized by proline but not by N‐acetylated sugars. Thapsigargin, an agent that inhibits uptake of Ca2+ into vesicles, sensitizes discharge. Discharge is sensitized upon incubating specimens in a caged analog of inositol 1,4,5‐trisphosphate (InsP3) and subsequently photoactivating it. Furthermore, following preincubation of specimens in certain low concentrations of caged InsP3 and subsequent photoactivation, lower concentrations of proline are required to maximally sensitize discharge. W7, an inhibitor of Ca2+/calmodulin (CaM), and KT5926, an inhibitor of CaM‐kinase II, reduce discharge sensitized by both N‐acetylated sugars and proline. Apparently, sugar receptors activate dihydropyridine‐sensitive Ca2+ channels, whereas proline receptors stimulate the production of InsP3, resulting in InsP3‐initiated release of Ca2+ from intracellular stores. This process may trigger Ca2+‐induced Ca2+ release from InsP3‐insensitive channels, which can be blocked by ryanodine or procaine. With either receptor, elevated intracellular Ca2+ binds calmodulin to form an active complex. CaM activates CaM‐kinase II, which, presumably, phosphorylates unidentified substrates, leading to sensitization of discharge. © 1995 Wiley‐Liss, Inc.
Do static electric forces contribute to the stickiness of a spider's cribellar prey capture threads?
Authors: Opell Brent D.
Published: June 2005

Cribellar thread is the most primitive type of prey capture thread produced by spiders. Its dry surface is formed of thousands of fine fibrils that catch on the setose surfaces of insects and, by an unknown mechanism, also hold smooth surfaces. Static electric attraction has been suggested as the force by which these smooth surfaces are held. However, when the stickiness of cribellar threads produced by Hyptiotes cavatus and Uloborus glomosus (family Uloboridae) was measured with contact plates that had similar textures but very different dielectric values, no support was found for this hypothesis. Differences in stickiness values were small and showed no relationship to the dielectric indexes of the surfaces used to measure stickiness. © 1995 Wiley‐Liss, Inc.
Interneurons involved in the control of multiple motor centers in crayfish
Authors: Burdohan John A. , Larimer James L.
Published: June 2005

A number of studies have suggested that abdominal positioning interneurons (APIs) in the crayfish Procambarus clarkii can influence activity in multiple motor centers. Previous work on this population of neurons has demonstrated that they control the activity of tonic abdominal motor neurons (MNs) which generate postural movements of the abdomen and, to some extent, the activity of swimmeret MNs. This investigation demonstrates that many identified APIs also affect the activity of two populations of MNs which open and close the uropods and, in some cases, that of the swimmeret MNs as well. The majority (64%) of APIs examined in this study have an excitatory effect on both populations of uropod MNs. A smaller number (23%) increase the level of activity in one population of uropod MNs, and suppress, or have no effect on, the activity of the other population of uropod MNs. Approximately 25% of the APIs which were examined influence the output of swimmeret MNs, in addition to affecting the activity of uropod MNs. There are also indications that previous estimates of the number of APIs may have been too low. This is based on the observation that many APIs possess what appear to be similar morphologies but generate different patterns of motor output. Taken together, these findings support the idea that APIs influence the output of multiple motor centers which play a role in the control of general body posture and balance in crayfish. © 1995 Wiley‐Liss, Inc.
Inhibition of Fundulus heteroclitus oocyte maturation in vitro by serotonin (5‐hydroxytryptamine)
Authors: Cerdá Joan , Petrino Teresa R. , Lin Yu‐Wai P. , Wallace Robin A.
Published: June 2005

The effects of serotonin (5‐hydroxytryptamine [5‐HT]) on oocyte maturation in vitro were tested on Fundulus heteroclitus full‐grown prematurational ovarian follicles. F.heteroclitus pituitary extract (FPE)‐ and steroid (17α,20β‐dihydroxy‐4‐pregnen‐3‐one [DHP])‐induced oocyte maturation were both inhibited by 5‐HT in a dose‐dependent and reversible manner. Maximum inhibition was observed at concentrations of 0.5 and 5 μM 5‐HT, respectively. The inhibitory action was also less effective when 5‐HT was combined with human chorionic gonadotropin than with FPE, which may indicate the presence of 5‐HT and/or other inhibitory substances in the pituitary extract. Inhibition by 5‐HT was independent of FPE‐induced steroid production by the follicle, since the levels of 17β‐estradiol and DHP released into the culture medium were not modified by the presence of 5‐HT. When follicles were preincubated with DHP for increasing times, subsequently washed, and transferred to steroid‐free, 5‐HT‐containing medium, inhibition by 5‐HT was also observed in follicles preincubated for up to 4 h; however, inhibition by 5‐HT was reduced or eliminated after 8 h or 16 h preincubation with DHP, respectively. These results suggest the presence of a putative 5‐HT receptor(s) that does not affect steroidogenic pathways in the ovarian follicle yet inhibits steroid‐induced oocyte maturation at some point downstream in the steroid transduction pathway of the oocyte. © 1995 Wiley‐Liss, Inc.
Characteristic features of preleptotene spermatocytes in Xenopus laevis: Increase in the nuclear volume and first appearance of flattened vesicles in these cells
Authors: Takamune Kazufumi , Teshima Kaori , Maeda Masayuki , Abé Shin‐Ichi
Published: June 2005

In Xenopus laevis, the spermatogenic cells derived from a primary spermatogonium divide synchronously and form a cyst surrounded by somatic Sertoli cells. To clarify how many times the spermatogonia divide mitotically before differentiating to primary spermatocytes, the number of spermatogenic cells in the cysts was counted. Each cyst which consisted of cells morphologically identical to secondary spermatogonia contained discrete numbers of cells such as 26, 27, or 28 cells. On the other hand, the number of the germ cells in a cyst of zygotene/pachytene primary spermatocytes was approximately 28. These results indicate that spermatogenic cells which had finished eight mitotic divisions and showed a similar morphology to secondary spermatogonia were preleptotene spermatocytes. The nuclei of the preleptotene spermatocytes increased in volume concomitantly with premeiotic DNA replication and differentiation into zygotene/pachytene primary spermatocytes. Therefore the cells morphologically indistinguishable from secondary spermatogonia but with larger nuclei than typical secondary spermatogonia were identified as preleptotene spermatocytes. Electron microscopic observations of the preleptotene spermatocytes identified as above showed that flattened vesicles, 0.1–0.5 μm in length, about 0.05 μm in width, and with more electron‐dense membrane than typical endoplasmic reticulum, first appeared in the cytoplasmic area of preleptotene spermatocytes. © 1995 Wiley‐Liss, Inc.
Changes in the pattern of osmoregulation in the brackish water amphipod Gammarus duebeni Lilljeborg (crustacea) during embryonic development
Authors: Morritt David , Spicer John I.
Published: June 2005

Eggs of the gammarid amphipod Gammarus duebeni Lilljeborg were cultured in sterilised in vitro conditions over a range of external seawater concentrations. The osmoregulatory ability of embryos and newly hatched individuals cultured in these conditions was examined over a wide range of external concentrations (75‐1,250 mOsm·kg−1 at 15°C). For all of the developmental stages examined the isosmotic point of the periembryonic fluid, P.F. (fluid from the space between the embryo and the vitelline membrane) or haemolymph from newly hatched individuals was similar (500‐550 mOsm·kg−1). Regulation of the P.F. by early stage 2 and 3 embryos, characterised by reddening cephalothorax and limb bud development, and of the haemolymph by new hatchlings followed a hyper‐isosmotic regulation pattern. In dilute external concentrations (between 50 and 100 mOsm·kg−1) P.F./haemolymph was maintained at a concentration of approx. 400 mOsm·kg−1): This difference diminished with increasing external concentration so that at and above 500‐550 mOsm·kg−1 P.F./haemolymph and external concentrations were isosmotic. Maintenance of a hyperosmotic P.F. in dilute media maintains cell function and establishes the osmotic gradient required for the uptake of water that results in the characteristic swelling of embryos. P.F. osmolality was regulated before the appearance of the coxal gills possibly via the vitelline membrane and/or the dorsal organ. In contrast, P.F. from later, stage 5‐7 embryos (eye pigmentation and cardiovascular development but still pre‐hatch) showed a distinct hyper‐hypo‐osmotic pattern of regulation: In dilute media the P.F. was maintained at levels between 400 and 550 mOsm·kg−1, and at external concentrations above 550 mOsm·kg−1 the P.F. was less concentrated than the medium (a difference of > 350 mOsm·kg−1 in the most concentrated external media). The functional significance of this hyper‐hypo‐osmoregulatory pattern is unknown although its appearance may be associated with the appearance of the coxal gills and the subsequent degeneration of the dorsal organ. This study has demonstrated that osmoregulatory function changes with ontogeny in amphipods and that the most “complicated” pattern (hyper‐hypo‐regulation) is associated with late embryo rather than the most “complex” (adult) stage. © 1995 Wiley‐Liss, Inc.
Acquisition of nerve dependence for the formation of a regeneration blastema in amputated hindlimbs of larval Xenopus laevis: The role of limb innervation and that of limb differentiation
Authors: Filoni Sergio , Velloso Cristiana Paranhos , Bernardini Sergio , Cannata Stefano M.
Published: June 2005

In larval and adult urodeles and late‐stage larval anurans, blastema formation after limb amputation requires an adequate nerve supply. Experimental evidence obtained from aneurogenic limbs indicates that, in urodeles, the acquisition of nerve dependence during embryonic development is due to the “addiction” of limb tissues to factors released by the ingrowing nerves rather than to limb differentiation. The aim of this work was to establish whether, in the toad Xenopus laevis, nerve‐dependence for blastema formation after hindlimb amputation, which is acquired gradually during larval development and becomes complete at stage 57 is due to limb innervation or to limb differentiation.

Two series of experiments were carried out. In the first series, limb differentiation was inhibited by treating the larvae with an anti‐thyroid drug, and innervation was maintained for an interval much longer than that normally required for development from nerve‐independent stages to stage 57. In the second series, the limb was caused to differentiate in the absence of nerves by maintaining the limbs denervated. Limb differentiation was often accelerated by treating early‐stage larvae with thyroxine or by grafting early‐stage limbs onto denervated limbs of late larvae, which, being near metamorphic climax, possessed high levels of circulating thyroid hormones.

Results showed that in the first series of experiments the denervated limbs formed regeneration blastemas after amputation, but in the second series they did not. It was therefore concluded that the acquisition of nerve dependence for blastema formation in larval Xenopus laevis is not directly imposed by factors released by the nerve fibers, but is strongly related to differentiation of limb tissues. © 1995 Wiley‐Liss, Inc.
Extrusion of microtubule doublet outer dense fibers 5‐6 associating with fibrous sheath sliding in mouse sperm flagella
Authors: Si Yuming , Okuno Makoto
Published: June 2005

Our previous experiments (Si and Okuno [1993a] Exp. Cell Res., 208:170–174) provided evidence that the fibrous sheath (FS) slid headward to middle piece in the activated mouse sperm flagellum when doublet microtubules together with their outer dense fibers (ODFs) extruded from the axoneme. Of the extruded doublet‐ODFs, however, which one was responsible for the FS sliding remained unresolved. The present study demonstrated that the FS sliding and the order of doublet‐ODFs extrusion in mouse sperm flagella were trypsin concentration dependent. Under the condition of mild trypsinization (0.1 μg/ml), only doublet‐ODFs 4, 5‐6 (doublet‐ODFs 5 and 6 were always paired), and 7 extruded from the axoneme. Furthermore, the extrusion of doublet‐ODFs 5‐6 was identified to precede doublet‐ODFs 4 and 7, and was considered the candidate responsible for FS sliding. In contrast, the high‐concentration trypsinization (4 μg/ml) led to extrusion of doublet‐ODFs 1, 2, and 9 following doublet‐ODFs 4, 5‐6, and 7. FS sliding, however, did not occur. © 1995 Wiley‐Liss, Inc.
Peptidergic and adrenergic innervation of the lachrymal gland in the euryhaline turtle, Malaclemys terrapin
Authors: Belfry C. Susan , Cowan F. Brian M.
Published: June 2005

The controlling factors of lachrymal gland secretions were examined in the euryhaline turtle, Malaclemys terrapin. Histochemical and immunocytochemical methods were used to localize some of the possible neurotransmitters involved.

There was no immunoreactivity to choline acetyltransferase, the enzyme synthesizing acetyl‐choline, nor did the histochemical technique for acetylcholinesterase produce positive results. Immunofluorescence and immunoperoxidase labels revealed vasoactive intestinal peptide (VIP)‐ and neuropeptide Y (NPY)‐like immunoreactivity in high concentrations surrounding the secretory tubules and ducts. Substance P produced a weak immunoreactivity in the interstitial space surrounding the ducts. Dopamine β‐hydroxylase, the enzyme synthesizing norepinephrine and epinephrine, was localized around the blood vessels. Immunogold labeling confirmed the presence of VIP‐ and NPY‐like reactivity in nerve varicosities close to the basement membrane of the secretory epithelium, and double‐labeling showed VIP and NPY are co‐localized within the same nerve terminals.

The results suggest that the secretory epithelium may be primarily under peptidergic control while the vascular system is under adrenergic control. This is possibly a new pattern of innervation for exocrine glands and may be related to the particular function of this salt gland in an euryhaline turtle. © 1995 Wiley‐Liss, Inc.
Proteins of Crustacean exoskeleton: IV. Partial amino acid sequences of exoskeletal proteins from the Bermuda land crab, Gecarcinus lateralis, and comparisons to certain insect proteins
Authors: Kumari S. Sindhu , Willis Judith H. , Skinner Dorothy M.
Published: June 2005

As in all decapod Crustacea, the exoskeleton of the land crab Gecarcinus lateralis consists of four layers. Prior electrophoretic analysis of proteins extracted from these layers revealed an abundance of small Mr proteins with acidic pIs as are found in insect cuticle (O'Brien et al. [1991] Biol. Bull., 181:427–441). Further, immunological cross‐reactivity between crab exoskeletal proteins and insect cuticular proteins has been demonstrated (Kumari and Skinner [1993] J. Exp. Zool., 265:195–210). Partial amino acid sequences of a number of proteins from the four exoskeletal layers are described here. Proteins were electrophoresed on two‐dimensional (2D) gels, transferred to polyvinylidene difluoride (PVDF) membranes, and stained; individual spots were recovered and their N‐termini were sequenced. In addition, a 14‐kDa protein (pI =5.4) from membranous layer (ML14) was eluted from 2D gels and digested with endoproteinase Lys‐C; N‐termini of its constituent peptides were sequenced.

The two epicuticular proteins differed from each other. Three proteins with identical electrophoretic mobility isolated from exocuticle, endocuticle, and membranous layer appeared to have identical N termini, while another electrophoretically identical set from the three layers appeared identical with each other but differed in three positions from the first set. Two proteins from the membranous layer both had a mass of 25 kDa but different isoelectric points. Their sequences were indistinguishable from each other but clearly distinct from another membranous layer protein. Another distinct sequence was found in a 14‐kDa protein from endocuticle, while a less acidic pair of 14‐kDa proteins from endocuticle and membranous layer were quite similar to one another. The three internal peptide fragments from ML14 were distinct, but one had regions similar to the ML14 N terminus. One crab exoskeletal protein sequence was similar to some structural proteins of vertebrates, whereas others had motifs found in insect cuticular proteins. The sequence similarities identified did not account for the antibody cross‐reactivity. © 1995 Wiley‐Liss, Inc.
A role for gene therapy in mental retardation
Authors: Moser Hugo W.
Published: June 2005

It is likely that gene therapy will be of most benefit in the single gene disorders that cause severe mental retardation. Reviews published in this issue focus on a number of these disorders in which vector development and preclinical trials or in vivo and ex vivo gene therapy are already in progress for urea cycle disorders, Lesch‐Nyhan disease, Gaucher disease, and phenylketonuria. On the other hand it is unlikely that gene therapy will hold much promise for the treatment of mild mental retardation which accounts for more than 80% of all cases of mental retardation. Here there is a strong environmental influence and early intervention and educational programs carry significant benefit. © 1995 Wiley‐Liss, Inc.
Gene therapy in mental retardation: Ethical considerations
Authors: Fletcher John C.
Published: June 2005

This article discusses resources in ethics for investigators who will pursue gene therapy for disorders that cause mental retardation: literature on the ethical principles that ought to guide clinical research, norms and practices of human experimentation, paradigm cases for human gene therapy, and professional integrity. I review major ethical considerations in the process of planning and conducting experiments, including benefits to subjects, the safety of subjects, voluntary informed assent of soem children and the permission of parents of children with retardation, as well as concerns for fairness in the selection of subjects. From a perspective of social justice, I criticize the potential of proposals for interventions to enhance mental abilities and argue for restraint and long‐range openness toward human germ‐line therapy to prevent genetic disorders. Depending on technical developments, gene therapy may provide humans with great benefits, including the prevention of the mental retardation caused by some genetic disorders. It is the responsibility of scientists and public review bodies to implement and monitor guidelines and safeguards so that human gene therapy can be fully tested and its benefits fairly distributed to those whose need and opportunity to benefit are greatest. © 1995 Wiley‐Liss, Inc.
Lesch‐Nyhan disease as a model for central nervous system directed gene therapy
Authors: Friedmann Theodore
Published: June 2005

As a model disease for the development of suitable gene transfer techniques and their physiological and neurological testing, Lesch‐Nyhan syndrome (LNS) provides important advantages, including the availability of a well‐characterized genetic defect and candidate metabolic targets, as well as a relevant animal model and appropriate gene transfer vectors. On the other hand, the serious gap between the level of characterization of the underlying genetic defects and the resulting neuropathological effects is one of several major impediments to the development of gene therapy. However, the dopamine defect that occurs in LNS may be much more amenable to genetic manipulation than is the deficiency of hypoxanthine guanine phosphoribosyltransferase. A genetic approach to the correction of this intermediate biochemical aberration, which probably is responsible for at least some neurological signs and symptoms of LNS, may be within reach of even the currently imperfect tools of gene therapy. Exploration of this possibility, along with other studies, is likely to lead, slowly but surely, to definition of the conditions under which the genetic and neurophysiological aberrations of LNS will be susceptible to a degree of correction that is not possible by drug therapy alone. © 1995 Wiley‐Liss, Inc.
Prospects for gene therapy in ornithine carbamoyltransferase deficiency and other urea cycle disorders
Authors: Robinson Michael B. , Batshaw Mark L. , Ye Xuehai , Wilson James M.
Published: June 2005

Although individually inborn errors of metabolism are rare, collectively they contribute significantly to morbidity and mortality in the pediatric age group. There are several reasons why, out of these inborn errors of metabolism, urea cycle disorders have emerged as potentially good candidates for the development of gene therapy. Studies have initially focused on ornithine carbamoyltransferase (OCT) deficiency in part because there are mouse models of this disease and in part because this disease is particularly resistant to current therapies. Both in vivo and ex vivo approaches to gene therapy are being developed for the treatment of urea cycle disorders. Ex vivo gene therapy is appealing because of the long‐term expression that can be achieved, but there are clear limitations to this approach. In vivo gene therapy using adenoviral vectors is attractive for several reasons, including the fact that the virus can be administered by intravenous injection, the high levels of expression observed after a single injection, and the rapidity of that expression. Studies of transgene expression in the mouse models of OCT deficiency (OCTD) have been encouraging, but have also provided evidence that the immune system may be involved in mediating two limiting aspects of this technology, transient gene expression and inflammation. Although deletions in adenoviral early genes should limit adenoviral late gene expression and subsequent viral replication, there is in vitro and in vivo evidence of late gene expression after infection with adenoviruses deleted of some of the early genes. Future studies will focus on systematically defining the components of the virus that are recognized by the immune system and mutating these gene products. The development of an approach to gene therapy that safely, stably, and efficiently transduces gene expression holds the promise of revolutionizing the treatment of inborn errors of urea synthesis. © 1995 Wiley‐Liss, Inc.
Gaucher disease: A tale of two species
Authors: McKinney Cindy E. , Sidransky Ellen , Lamarca Mary E. , Riviere Isabelle , Holleran Walter M. , Martin Brian M. , Willemsen Rob , Mulligan Richard C. , Ginns Edward I.
Published: June 2005

Gaucher disease, the result of inherited deficiency of glucocerebrosidase, is the most common lysosomal storage disease. No naturally occurring animal models are available for study of its pathophysiology or the development of new therapeutic strategies such as gene therapy. We describe an approach to gene targeting in embryonic stem cells to “knock out” glucocerebrosidase activity and generate a mouse line having the phenotype of severely affected infants with type 2 Gaucher disease. We also discuss an experiment in gene therapy using lethally irradiated mice that are reconstituted with murine fetal liver hematopoietic cells transduced with retrovirus encoding human glucocerebrosidase. Analyses of these animals show production of active human glucocerebrosidase in lysosomes of macrophages for at least 12 months after transplantation. © 1995 Wiley‐Liss, Inc.
Functional assessment and intervention in community settings
Authors: Sprague Jeffrey R. , Horner Robert H.
Published: June 2005

Functional assessment has emerged as an important strategy for designing effective interventions for serious problem behavior. A comprehensive functional assessment identifies the behavior of concern, stimuli correlated with the onset and maintenance of the behavior, features of the setting in which the behavior occurs, and behaviors (typically communication) that may serve to replace the problem behavior. We present a historical and modern context for functional assessment and, in juxtaposition to this, discuss the purposes of functional assessment as a tool for researchers and practitioners. We review the current range of functional assessment applications, emphasizing the strengths and limitations of each; propose needed extensions of existing functional assessment methods; and discuss the critical role of functional assessment in designing effective community‐based behavioral interventions. © 1995 Wiley‐Liss, Inc.
Bio‐behavioral diagnosis and treatment of self‐injury
Authors: Mace F. Charles , Mauk Joyce E.
Published: June 2005

Self‐injurious behavior(SIB) in individuals with mental retardation is a severe behavioral disorder that has significant social and medical consequences. Although considerable research evidence indicates that SIB can be maintained by either environmental contingencies or biologic conditions, integrative diagnostic and treatment models have yet to be developed. We have piloted a bio‐behavioral diagnostic and treatment model to classify and subtype cases of SIB, leading to the selection of specific behavioral or pharmacologic treatments. A functional analysis defines SIB as operant, possibly biologic, or mixed operant and possibly biologic. Subtyping within these classes is based on the delineation of specific operant functions and the clinical characteristics of the individual patient. Our preliminary research suggests that this empirical decision‐making model can lead to specific treatments that are effective and that reduce the incidence of nonresponders to behavioral intervention and medication. © 1995 Wiley‐Liss, Inc.
Opiate mechanisms in self‐injury
Authors: Sandman Curt A. , Hetrick William P.
Published: June 2005

Self‐injuring behavior is among the most unmanageable, expensive, destructive, and unpredictable behaviors exhibited by human beings. Interventions that included punishment or restraint were among the most successful for acute control of SIB. Both painful shock and restraint stimulate release of the body's own opiates into the bloodstream. These powerful agents, which possess analgesic and addictive properties, are more potent than morphine. Based on these observations, two versions of the opiate hypothesis have evolved to explain SIB. The analgesia hypothesis suggests that high‐circulating levels of β‐endorphin (βE) in individuals with SIB reduces the perception of pain. With the experience of pain reduced, individuals inflict self‐harm as a form of stimulation. The addiction hypothesis presumes that the release of opiates after “pain” or SIB produces pleasure. As a consequence, individuals exhibiting SIB become addicted to their own opiate system. The strongest link between SIB and the opiate hypotheses is the finding that opiate receptor blockers attenuate and sometimes eliminate this behavior. In addition to providing a possible treatment for some SIB individuals, these findings suggest that a specific opiate system, βE and mu receptor, is disregulated (by elevated levels of opiates, abnormally sensitive opiate receptors, or uncoupling of co‐released peptides). Recent studies have found that βE was elevated after an SIB episode, but that co‐released peptides such as ACTH were not. Selective release of βE after SIB provides evidence that endogenous opiates have a direct association with SIB. This association supports speculation that endogenous opiates maintain SIB. © 1995 Wiley‐Liss, Inc.
Molecular and clinical correlations in fragile X syndrome
Authors: Hagerman Randi Jenssen
Published: June 2005

Since the advent of DNA testing after 1991, variations in molecular patterns, such as the CGG expansion length, mosaicism, partial methylation, and X‐inactivation, have been correlated with the degree of phenotypic involvement in fragile X syndrome. Although some reports conflict it appears that a low CGG repeat number within the full mutation range or lack of methylation, whether almost complete or partial, is associated with only mild involvement in males. A subgroup of individuals with the premutation may also demonstrate mild problems associated with fragile X syndrome. This article reviews preliminary studies of FMRP expression and discusses a hypothesized correlation between FMRP expression and phenotypic involvement. Molecular and clinical correlations are advancing our understanding of the mild range of phenotypic involvement in fragile X syndrome, which involves learing disabilities and emotional difficulties, but not mental retardation. © 1995 Wiley‐Liss, Inc.
An animal model for fragilex syndrome
Authors: Willems Patrick J. , Reyniers Edwin , Oostra Ben A.
Published: June 2005

Although the fragile X gene (FMR1) was isolated more than four years ago, little is known about its physiologic function and the pathological mechanisms that lead to the abnormalities observed in fragile X patients. To provide more insight into the physiologic role of FMR1, we have developed a transgenic mouse with a loss‐of‐function (Knockout) mutation in the FMR1 gene using homologous recombination in embryonic stem cells. The mutant mice lack normal FMR1 mRNA and protein, as is also the case in human fragile X syndrome. The mice show enlarged testes and impaired performance in cognitive function tests, a clinical picture very similar to that of human patients. As a consequence, this animal model might be useful in elucidating the physiologic function of FMR1 and the pathological mechanisms that lead to mental retardation and behavioral abnormalities in fragile X syndrome. Experimental drug treatment can now be tried to ameliorate the congnitive function and behavior of affected mice and, if these experiments are successful, human trials can be initiated. Furthermore, gene therapy experiments by introduction of a foreign FMR1 gene into mutant mice can now be begun. © 1995 Wiley‐Liss, Inc.
Pathogenesis of cleft lip and its relation to embryonic face shape in A/J and C57BL mice
Authors: Trasler Daphne G.
Published: June 2005

A comparison was made of early face embryology in the inbred A/J mouse strain with a spontaneous frequency of about 12% cleft lip and the C57BL/6J strain which virtually never has cleft lip. From the time of their appearance the medial nasal, lateral nasal and maxillary processes are connected by an isthmus. Subsequently there is fusion between the adjacent epithelia of medial and lateral nasal processes. Failure of this fusion, followed by breakdown of the isthmus, results in cleft lip. Just before and when the adjacent epithelia of medial nasal and lateral nasal processes meet and fuse at the posterior end of the nasal pit, the medial nasal processes of A/J embryos were more prominent, more medially placed, and diverged less than those of C57BL/6J. It is suggested that (a) cleft lip is a threshold character or quasi‐continuous variant such that a very slight change in the divergence of the medial nasals leads to their partial or complete lack of fusion with the lateral nasals. This is followed by a lack of fusion of medial and maxillary processes, and a complete or partial breakdown of the isthmus, resulting in a cleft lip; (b) the strain differences in type of aspirin‐induced cleft lip and in topography and growth of the facial processes between the two strains are causally related to the A/J predisposition to cleft lip.