bluuebunny/crossref_metadata_2025 huggingface dataset:
Endothelium‐Derived Hyperpolarizing Factor: Is There A Novel Chemical Mediator?
Authors: Triggle Chris R , Ding Hong
Published: March 2003

1. Endothelium‐derived hyperpolarization (EDH) has been reported in many vessels and an extensive literature suggests that a novel, non‐nitric oxide and non‐prostanoid, endothelium‐derived factor(s) may be synthesized in endothelial cells.

2. The endothelium‐dependent hyperpolarizing factor, or EDHF, is synthesized by the putative EDHF synthase and mediates its cellular effects by either, directly or indirectly, opening K channels on vascular smooth muscle cells or, via hyperpolarization of the endothelial cell, by facilitating electrical coupling between the endothelial and the vascular smooth muscle cell.

3. The question of the chemical identity of EDHF has received considerable attention; however, no consensus has been reached. Tissue and species heterogeneity exists that may imply there are multiple EDHF. Leading candidate molecules for EDHF include an arachidonic acid product, possibly an epoxygenase product, or an endogenous cannabinoid, or simply an increase in extracellular K+.

4. An increasing body of evidence suggests that EDH, notably in the resistance vasculature, may be mediated via electrical coupling through myoendothelial gap junctions and the existence of electrical coupling may negate the need to hypothesize the existence of a true endothelium‐derived chemical mediator.

5. In this paper we review the evidence that supports and refutes the existence of a novel EDHF versus a hyperpolarization event mediated solely by myoendothelial gap junctions.
Pharmacological Profile Of Nociceptin/Orphanin Fq Receptors
Authors: Calo’ Girolamo , Rizzi Anna , Bigoni Raffaella , Guerrini Remo , Salvadori Severo , Regoli Domenico
Published: March 2003

1. Nociceptin/orphanin FQ (NC) and its receptor (OP4) represent a novel peptide/receptor system pharmacologically distinct from classical opioid systems.

2. Via OP4 receptor activation, NC regulates several biological functions,
both at peripheral and central levels; therefore, the OP4 receptor may
be viewed as a novel target for drug development. However, the pharmacology of this receptor is still in its infancy, with few molecules interacting selectively with this receptor.

3. In the present article, we review the findings of studies that have investigated the pharmacological profile of ligands selective for the OP4 receptor,
these being two antagonists, the peptide [Nphe1]NC(1–13)NH2
and the non‐peptide J‐113397, and two agonists, the peptide [Arg14,Lys15]NC, and the non‐ peptide Ro 64‐6198.

4. The results of these studies indicate that agents that selectively activate or block the OP4 receptor may represent new potential drugs for the treatment of human diseases.
Modulation Of [35S]‐Tert‐ Butylbicyclophosphorothionate Binding By Somatostatin In Rat Hypothalamus
Authors: Chigr F , M’hamed S Ba , Najimi M
Published: March 2003

1. The present study was designed to assess the effect of the tetradecapeptide somatostatin on the GABAA receptor complex in the rat hypothalamus.

2. GABAA receptors were labelled with [35S]‐tert‐butylbicyclophosphorothionate
(TBPS), which binds in or near the chloride channel, and binding as assessed by in
vitro quantitative autoradiography using a computer‐assisted image analysis system.

3. Somatostatin inhibited the binding of [35S]‐TBPS to the convulsant
site of the hypothalamic GABAA receptor complex of rat slide‐mounted hypothalamic
structures in a concentration‐dependent manner with an affinity in the micromolar
range (10–6 to 3 × 10–6 mol/L).
Somatostatin appeared to mimic the effects of the neurosteroid 5α‐pregnane‐3αol‐one
(5α3αP), GABA and picrotoxin on [35S]‐TBPS binding
in the rat hypothalamus in all structures examined. Furthermore, GABA or muscimol
(a GABAA receptor agonist), when added to the incubation medium, enhanced
the capacity of somatostatin to inhibit [35S]‐TBPS binding, with an IC50
of 10–7 mol/L. However, incubation with bicuculline (a GABAA
receptor antagonist) led to the abolition of the inhibitory effect of somatostatin
on [35S]‐TBPS specific binding in rat hypothalamus.

4. The present results demonstrate the presence of a modulatory effect of somatostatin on the GABAA receptor complex in rat hypothalamic structures. Furthermore,
the data suggest that somatostatin allosterically modifies [35S]‐TBPS binding through a mechanism similar to that of GABA. Taken together, these results provide evidence for the presence of somatostatin– GABA interactions in rat hypothalamus.
Role Of CA2+‐Activated K+ Channels In The Regulation Of Basilar Arterial Tone In Spontaneously Hypertensive Rats
Authors: Kamouchi Masahiro , Kitazono Takanari , Nagao Tetsuhiko , Fujishima Masatoshi , Ibayashi Setsuro
Published: March 2003

1. Ionic channels appear to play an important role in contractile responses of the cerebral arteries and, thereby, contribute to the regulation of cerebral circulation. In the present study, we investigated the role of large‐conductance Ca2+‐activated
K+ (BKCa) channels in the regulation of cerebral arterial tone during chronic hypertension.

2. Ring segments of the basilar artery from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto (WKY) rats were placed in bath chambers and the isometric tension of each ring was measured.

3. Application of inhibitors of BKCa channels, namely tetraethylammonium (TEA; ≥ 0.1 mmol/L) and charybdotoxin (CTX; ≥ 0.1 nmol/L), produced spontaneous contraction with rhythmic oscillation in the basilar artery from SHR.

4. The oscillatory contraction was not induced by 5‐hydroxytryptamine (0.01–10 μmol/L) or depolarization by external high K+ (20–60 mmol/L).

5. The rhythmic contraction was completely abolished by either the removal of external Ca2+ or the application of nicardipine (10 nmol/L).

6. The oscillation was not affected by the substitution of external Cl– by various equimolar anions (i.e. acetate, benezenesulphonate, bromide and isethianate).

7. The amplitude of the oscillation was dose‐dependently increased by the vasodilators forskolin and sodium nitroprusside, as well as by stimulation of the endothelium with histamine and acetylcholine, whereas the frequency was decreased.

8. In contrast, the oscillation was eliminated by depletion of Ca2+ stores by caffeine. Neither TEA (10 mmol/L) nor CTX (10 nmol/L) produced any significant contraction of the basilar artery in WKY rats.

9. These results suggest that BKCa channels may play an important role in regulating the resting tone of the cerebral artery in SHR.
Effects Of Melanocortins On Cardiovascular Regulation In Rats
Authors: Ramaekers D , Beckers F , Demeulemeester H , Bert C , Denef C , Aubert AE
Published: March 2003

1. Of the melanocortin peptides, γ2‐melanocyte‐stimulating hormone (MSH) has been attributed a cardiovascular effect, inducing an increase in blood pressure and heart rate. Although still controversial, this effect, based on pharmacological blockade experiments, is supposed to be mediated through sympathetic activation.

2. The aims of the present study were to identify the N‐terminal pro‐opiomelanocortin (N‐POMC) fragments and melanocortins that influence blood pressure and heart rate and to investigate the real‐time changes in baroreflex sensitivity and in sympathetic and vagal modulation underlying cardiovascular effects in conscious rats without the use of pharmacological blockade.

3. Intracerebroventricular administration of different melanocortins and N‐POMC induced a long‐lasting dose‐ dependent pressor response from 1 nmol onwards, with only a small initial bradycardic response with the highest dose.

4. Coinciding with this pressor response, an elicitation of the low‐frequency (LF) component was observed in spectral analysis of both blood pressure variability (BPV) and heart rate variability (HRV), followed by the high‐frequency (HF) component in at least BPV. Baroreflex sensitivity remained unchanged.

5. After intravenous administration, γ2‐MSH produced a short‐lasting dose‐dependent pressor and cardioaccelerator response with very rapid onset with concentrations from 1 nmol onwards.

6. Continuous infusion of γ2‐MSH depressed baroreflex sensitivity and simultaneously increased both components of BPV, with a radical reduction of the LF component and a preserved vagal HF component in HRV.

7. Of all the intravenously administered melanocortins, only γ2‐MSH
was active. The central effect is likely to depend on an increase of (α‐)sympathetic outflow.

8. For the peripheral effect, γ2‐MSH appeared to act as a baroreceptor reflex‐blocking agent, being compatible with a role in the acute stress response.
A Rat Model Of Ischaemic Or Dilated Cardiomyopathy For Investigating Left Ventricular Repair Surgery
Authors: Nishina Takeshi , Miwa Senri , Yuasa Sadatoshi , Nishimura Kazunobu , Komeda Masashi
Published: March 2003

1. The effects of left ventricular repair (LVR) surgery for ischaemic or dilated cardiomyopathy are not fully understood. The development of a proper animal model will help to resolve this issue.

2. The ischaemic cardiomyopathy (ICM) model used was as follows. Twenty‐six rats developed ICM with a large akinetic left ventricular (LV) area after ligation of the left anterior descending artery (LAD). Four weeks after surgery, 13 rats underwent LVR by placating the akinetic area (LVR group), while 13 underwent rethoracotomy alone (sham group).

3. The dilated cardiomyopathy (DCM) model is as follows. Six Dahl salt‐sensitive (DS) rats were fed an 8% NaCl diet from the age of 9 weeks and developed DCM. These rats had LVR by plicating the LV area between the papillary muscle bases (LVR group), while other rats underwent rethoracotomy alone (sham group).

4. Before LAD ligation in the ICM model or starting the high‐salt diet in the DCM model and just before and after LVR or sham surgery, LV dimensions were measured by echocardiography and the heart rate and systolic blood pressure were recorded by the tail‐cuff method.

5. In the ICM model, all rats survived the second surgery, after which LV end‐diastolic diameter (EDD) decreased, LV fractional shortening (FS) increased (both P < 0.001 vs sham) and heart rate increased (P < 0.05) in the LVR group.

6. In the DCM model, LV EDD decreased and LV FS increased (both P < 0.001) in the LVR group.

7. Both models developed LV dilatation, tolerated LVR and enable reproducible physiological evaluation of the LV. Because the rats survived thereafter, both models may provide a useful tool for various investigations.
Antihyperglycaemic And Anti‐Oxidant Properties Of Anoectochilus Formosanus in Diabetic Rats
Authors: Shih Chun‐Ching , Wu Yueh‐Wern , Lin Wen‐Chuan
Published: March 2003

1. In the present study, we investigated aqueous extracts of Anoectochilus formosanus (AFE) for antihyperglycaemic and anti‐oxidant effects in diabetic rats induced by streptozotocin (STZ).

2. Diabetic rats were randomly divided into groups and treated orally by gavage with vehicle (distilled water) or AFE (1 and 2 g/kg), once a day for 21 days.

3. At the end of the 21 day period, AFE (2 g/kg) significantly reduced fasting blood glucose, serum fructosamine, triglycerides and total cholesterol compared with vehicle‐treated diabetic rats. In vehicle‐treated diabetic rats, levels of renal lipid peroxidation were increased, whereas glutathione concentrations were not affected. Renal lipid peroxidation levels were significantly lower and renal reduced glutathione (GSH) concentrations were significantly higher in AFE‐treated diabetic rats compared with vehicle‐treated diabetic rats. The diabetic kidney in the vehicle‐treated group showed a decrease in catalase, but the activity of glutathione peroxidase (GSH‐Px) was increased.

4. The activity of catalase, but not GSH‐Px, was significantly reversed by AFE treatment. These results indicate that AFE (1 and 2 g/kg) not only possesses an antihyperglycemic effect, but that it may also reduce oxidative stress in diabetic rats.
Molecular moulds with multiple missions: Functional sites in three‐finger toxins
Authors: Kini R Manjunatha
Published: March 2003

1. Snake venoms are complex mixtures of pharmacologically active peptides and proteins.

2. These protein toxins belong to a small number of superfamilies of proteins. The present review describes structure–function relationships of three‐finger toxins.

3. All toxins share a common structure of three β‐stranded loops extending from a central core. However, they bind to different receptors/acceptors and exhibit a wide variety of biological effects.

4. Thus, the structure–function relationships of this group of toxins are complicated and challenging.

5. Studies have shown that the functional sites in these ‘sibling’ toxins are located on various segments of the molecular surface.
Effects of creatine loading and depletion on rat skeletal muscle contraction
Authors: Gagnon M , Maguire M , MacDermott M , Bradford A
Published: March 2003

1. In humans, the effects of dietary creatine supplementation are controversial, with some studies showing increased muscle force and fatigue resistance and others reporting no effect on exercise performance. Little is known about the effects of creatine on muscle contractile properties.

2. Rats were fed a standard diet, creatine for 10 days or β‐guanidinopropionate, which depletes muscle creatine, for 7 days. Contractile properties were measured in isolated extensor digitorum longus and sternohyoid muscle as representative limb and upper airway dilator muscles, respectively.

3. Creatine had no effect on specific twitch and tetanic tension, contractile kinetics, twitch/tetanus tension ratio, the tension–frequency relationship or fatigue in both muscles. β‐Guanidinopropionate had no effect on the twitch and tetanic tension, contractile kinetics, twitch/tetanus tension ratio or tension–frequency relationship, but significantly increased (P < 0.05, anova) fatigue in both muscles.

4. Therefore, although creatine depletion increases fatigue, creatine loading has no effects on extensor digitorum longus and sternohyoid muscle contractile properties.
Antiplatelet effects of policosanol (20 and 40 mg/day) in healthy volunteers and dyslipidaemic patients
Authors: Arruzazabala María Lourdes , Molina Vivian , Mas Rosa , Fernández Lilia , Carbajal Daisy , Valdés Suria , Castaño Gladys
Published: March 2003

1. The present study was undertaken to compare the effects of a higher dose of policosanol, a cholesterol‐lowering drug, (40 mg/day) with the effects of 20 mg/day policosanol on platelet aggregation in healthy volunteers and type II hypercholesterolaemic patients.

2. Study subjects were randomized to receive, under double‐blind conditions, placebo or policosanol (20 or 40 mg/day) for 30 days once a day. Blood sampling was performed at baseline and after 30 days on therapy.

3. Platelet aggregation was induced with three aggregating agents: arachidonic acid (AA), collagen and low doses of ADP.

4. Policosanol (20 and 40 mg/day) moderately yet significantly reduced platelet aggregation, but no differences were observed in the effects produced by either dose of policosanol. In healthy volunteers, policosanol at 20 and 40 mg/day inhibited aggregation induced by 2 mmol/L AA (28.2 and 24.9%, respectively), 1 µg/mL collagen (21.1 and 20.2%) and 1µmol/L ADP (30.9 and 29.1%). Changes that occurred following the adminstration of placebo were not significant, although an upward trend for collagen‐ and ADP‐induced aggregation occurred in normal and hypercholesterolaemic subjects, respectively, thus partially masking the effects of policosanol on these responses.

5. The antiplatelet effects of policosanol at 20 and 40 mg/day in hypercholesterolaemic patients were also similar, so that both doses inhibited aggregation induced by 1.5 mmol/L AA (20.1 and 33.0%, respectively), 0.5 µg/mL collagen (22.7 and 21.1%) and1 µmol/L ADP (40.5 and 34.7%).

6. In addition, after 30 days of therapy, 20 and 40 mg/day policosanol significantly (P < 0.01) reduced low‐density lipoprotein–cholesterol (15.9 and 17.0%, respectively) and total cholesterol (12.4 and 12.3%, respectively; P < 0.05), yet increased high‐density lipoprotein–cholesterol values by 5% in both groups (P < 0.05).

7. Triglycerides were decreased compared with baseline, but not with respect to the placebo.

8. We conclude that the antiplatelet effects induced by 40 mg/day policosanol administered for 30 days to healthy volunteers and to hypercholesterolaemic patients were similar to the effects induced by 20 mg/day policosanol. Thus, no enhancement of the response was achieved with the use of a higher dose of policosanol in study patients.
Effects of Ginkgo biloba extract (EGb 761) on cerebral thrombosis and blood pressure in stroke‐prone spontaneously hypertensive rats
Authors: Sasaki Yasuto , Noguchi Takanori , Yamamoto Etsuko , Giddings John C , Ikeda Katsumi , Yamori Yukio , Yamamoto Junichiro
Published: March 2003

1. An extract of Ginkgo biloba (EGb 761) has been reported to alleviate cerebrovascular problems. In the present study, we investigated the antithrombotic effects of EGb 761 in cerebral blood vessels of stroke‐prone spontaneously hypertensive rats (SHRSP/Izm).

2. In the present study, EGb 761 was administered orally to SHRSP/Izm at 60 and 120 mg/kg each day for 3 weeks from the age of 7 weeks. The age‐related increase in blood pressure observed in SHRSP was suppressed significantly by EGb 761 at both doses 3 weeks after treatment.

3. Thrombotic potential was assessed in vivo using a He‐Ne laser‐induced thrombosis model and was significantly suppressed by EGb 761.

4. The anti‐oxidant effects of EGb 761 were determined by measurement of urinary 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG). At 120 mg/kg, EGb 761 decreased 8‐OHdG significantly compared with control animals.

5. Urinary nitrite/nitrate, nitric oxide (NO) metabolites, were increased significantly after administration of EGb 761. Expression of endothelial NO synthase (eNOS) mRNA was measured using a real‐time quantitative reverse transcription–polymerase chain reaction method. The expression of eNOS mRNA in the EGb 761 group (120 mg/kg) was significantly higher than in the control group.

6. The results indicate that EGb 761 decreases blood pressure and mediates strong antithrombotic and anti‐oxidant effects in SHRSP. These pharmacological activities may contribute to the beneficial properties of EGb 761 observed in clinical practice.
Evolution of drug metabolism: Hitchhiking the technology bandwagon
Authors: Miners John O
Published: March 2003

1. The application of a range of established and emerging technologies and experimental approaches has allowed investigation of cytochrome P450 (CYP) and uridine diphosphate‐glucuronosyltransferase (UGT) at the functional, structural and molecular levels to address questions of therapeutic relevance, particularly the wide interindividual variability in metabolic clearance characteristic of drugs and chemicals metabolized by these enzymes.

2. Studies in vivo initially identified the various factors that contribute to interindividual variability. Subsequently, human liver microsomal kinetic approaches, together with the cloning and functional characterization of recombinant CYP and UGT isoforms, led to the development of in vitro strategies that allowed the qualitative prediction of those factors likely to alter the metabolic clearance of a given compound in vivo. More recently, computer (in silico) modelling has been used to complement the laboratory based procedures.

3. The application of molecular biological approaches additionally allowed identification of the mutations responsible for CYP and UGT genetic polymorphism and, in some instances, the domains and individual amino acids that confer isoform substrate and inhibitor selectivities. Homology models, developed using X‐ray crystallographic data as the template, potentially enable prediction of the functional consequences of altered CYP structure.

4. The rapid advances occurring in genomics, proteomics, gene expression analysis and computer modelling will allow further unravelling of the complexities of drug metabolism and improved prospects for the individualization of drug therapy.
Changes in mean arterial pressure predict degranulation of renomedullary interstitial cells
Authors: Maric Christine , Harris Peter J , Alcorn Daine
Published: March 2003

1. Renomedullary interstitial cells (RMIC) are characterized by numerous intracellular granules thought to contain renal medullary antihypertensive substances. However, the nature of the trigger for RMIC degranulation remains to be elucidated. The present study examines the effects of acute alterations in mean arterial pressure (MAP) and medullary blood flow (MBF) on RMIC granulation.

2. Basal MAP and MBF in anaesthetized Sprague‐Dawley rats (n = 4/group) were altered by intravenous infusions of vasoactive agents, including angiotensin II alone or with a nitric oxide (NO) synthase inhibitor (Nω‐nitro‐l‐arginine) or NO donor (sodium nitroprusside), noradrenaline and by carotid artery clamping. Following these treatments, kidneys were examined by electron microscopy and the absolute volume of granules in the renal medulla was calculated using unbiased stereological methods.

3. Acute increases in MAP, regardless of the treatment causing the increase, were associated with a reduction in the absolute volume of granules in the range of 42–67%. Regression analysis revealed that only increases in MAP, but not MBF, strongly predict RMIC degranulation.

4. Despite previous reports that changes in MBF activate renomedullary antihypertensive activity, we conclude that the change in MAP is an important determinant of the activity of the blood pressure‐lowering mechanism of the renal medulla, with the assumption that the medullary lipids mediate the antihypertensive property of the renal medulla.
Sperm surface heparin/heparan sulfate is responsible for sperm binding to the uterine envelope in the newt, Cynops pyrrhogaster
Authors: Nakai Satoshi , Watanabe Akihiko , Onitake Kazuo
Published: March 2003

The sperm‐binding properties of egg envelopes are investigated in the newt, Cynops pyrrhogaster. Sperm binding was only seen on the uterine envelope when acrosome‐reacted sperm were inseminated. No acrosome‐intact sperm bound to the envelopes. By scanning electron microscopic observation, acrosome‐reacted sperm were revealed to bind to a seat‐like structure present on the surface of the uterine envelope. Sperm binding to the uterine envelope was inhibited by treatment of eggs with heparin or heparan sulfate, or treatment of acrosome‐reacted sperm with heparinase prior to insemination. A molecule with a molecular mass of 75 kDa was purified from the uterine envelope by affinity chromatography with heparin‐Sepharose. These results indicated that sperm binding was mediated by heparin‐like molecules expressed on the surface of acrosome‐reacted sperm and the 75 kDa molecule was present as a constituent of uterine envelopes.
Expression of a src‐type protein tyrosine kinase gene, AcSrc1, in the sea urchin embryo
Authors: Onodera Hiroshi , Kobari Kazuki , Sakuma Makoto , Sato Miki , Suyemitsu Takashi , Yamasu Kyo
Published: March 2003

By screening a cDNA library and 3′‐rapid amplification of cDNA ends, the cDNA for a non‐receptor type protein tyrosine kinase from the sea urchin Anthocidaris crassispina was analyzed. The deduced protein (AcSrc1) with the highest identity of about 60% to mammalian Src family kinases shows the characteristic features of the Src family. AcSrc1 mRNA is maternally expressed in unfertilized eggs, while zygotic expression is first detected in blastulae and continues through the pluteus stage. Zygotic mRNA expression, visualized by in situ hybridization, is detected specifically in archenteron at the gastrula stage, while it is restricted in plutei to the midgut and hindgut, suggesting specific roles for AcSrc1 in the formation and/or functions of the digestive tract. Meanwhile, western blot analysis has shown that the AcSrc1 protein is constantly expressed throughout embryogenesis. By immunostaining, it was found that the protein (distributed evenly in the cytoplasm of unfertilized eggs) is translocated to the membrane after fertilization. All through the following development, AcSrc1 was localized to the peripheries of different embryonic cells, although at a relatively low level of localization at the boundaries between adjacent cells.
Degradation of yolk platelets in the early amphibian embryo is regulated by fusion with late endosomes
Authors: Komazaki Shinji , Hiruma Tamiko
Published: March 2003

The eggs of many animal species contain a large store of yolk platelets, lipid droplets and glycogen granules; these are consumed during early embryogenesis. However, the mechanisms by which degradation of these stored materials occurs during early embryogenesis are not clearly understood. The mechanisms underlying yolk degradation in amphibian (newt) embryos were investigated. Electron microscopy using an anion marker, cationic ferritin, revealed that yolk platelets were degraded after fusion with late endosomes containing primary lysosomes. Electron microscopy and the results of experiments using a number of reagents with selective effects on intracellular transport suggested that yolk degradation activity in early amphibian embryos may be regulated at the point of fusion between late endosomes and yolk platelets.
Transplantation of blastula nuclei to non‐enucleated eggs in the medaka, Oryzias latipes
Authors: Niwa Katsutoshi , Ladygina Tatiana , Kinoshita Masato , Ozato Kenjiro , Wakamatsu Yuko
Published: March 2003

Studies of nuclear transplantation were conducted to establish methods for the production of clones of fish, using a small laboratory fish, medaka, Oryzias latipes. As the first step of the study, single‐blastula nuclei of an inbred strain with the wild‐type body color were transplanted into non‐enucleated unfertilized eggs of an outbred orange–red strain. Of 845 operated eggs, 45 hatched into fry exhibiting the wild‐type body color, one of the donor markers. Twenty‐seven of these nuclear transplants grew to the adult stage and clearly exhibited external secondary sexual characteristics. Fourteen were females and 13 were males. The allozyme analysis of phosphoglucomutase, measurements of relative DNA content by microfluorometry and chromosome counts consistently indicated that the nuclear transplants were triploids that originated from both the diploid donor nuclei and the haploid recipient pronuclei. In the crossing experiments between the nuclear transplants and the orange–red strain, most of the male nuclear transplants were sterile, whereas one male produced a viable offspring with wild‐type body color. All of the female nuclear transplants were sterile. Macroscopic observations of their gonads showed that the testes appeared normal and the ovaries appeared degenerated. These features of the reproductive potential and the morphology of gonads also indicated that the nuclear transplants were triploids. These results demonstrated that a basic technique for nuclear transplantation in medaka was established.
Newt RAD51: Cloning of cDNA and analysis of gene expression during spermatogenesis
Authors: Yamamoto Takashi , Hikino Taketoshi , Nakayama Yuki , Abé Shin‐Ichi
Published: March 2003

A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated. The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95–96% amino acid identity to Xenopus and mammalian RAD51. Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain. In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (≥ eight cells in a cyst), reaching a maximum level in leptotene–zygotene spermatocytes, and thereafter declined. These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.
cDNA cloning of a stage‐specific gene expressed during HCG‐induced spermatogenesis in the Japanese eel
Authors: Miura Chiemi , Miura Takeshi , Kudo Nozomi , Yamashita Masakane , Yamauchi Kohei
Published: March 2003

A single injection of human chorionic gonadotropin (HCG) can induce complete spermatogenesis in immature Japanese eel (Anguilla japonica) testes consisting of only premitotic spermatogonia. Proliferation of spermatogonia, meiosis and spermiogenesis begin on 3, 12 and 18 days after HCG injection, respectively. To isolate the genes responsible for regulating the initiation of meiosis, differential mRNA display using poly (A)+ RNA extracted from testes of eels at different times after HCG treatment was carried out. Five cDNA clones in which expression was initiated before the onset of meiosis were obtained. Northern blot analysis showed that one clone, which encoded activin βB subunit, was expressed in the initial phase of spermatogenesis (1–6 days after HCG treatment), in agreement with the previous suggestion that activin B induces the initiation of spermatogenesis in the Japanese eel. The remaining four were expressed in the testes during the following time frames: 3–18 days (two clones), 6–18 days (one clone) and 9–18 days (one clone) after HCG treatment. One of the two clones expressed on day 3 exhibited strong expression on days 12 and 15, just at the initiation period of meiosis. This clone was selected as a candidate gene responsible for initiating meiosis, and its full‐length cDNA isolated. The cDNA contained an open reading frame of 1571 nucleotides encoding a protein of 260 amino acid residues, which showed high homology with the proliferating cell nuclear antigen (PCNA) of human, mouse and Xenopus. Northern blot analysis using eel PCNA cDNA showed that a 1.6 kb transcript first appeared on day 3 and became abundant, reaching maximum levels on days 12–15. In situ hybridization analysis revealed that PCNA mRNA was expressed strongly in late type B spermatogonia before the sixth mitotic division. It has already been shown that spermatogonia have a regulatory point to enter meiosis between the fifth and sixth mitotic division. The coincidence of PCNA expression and this regulatory point suggests an involvement of PCNA in the progression of mitotic germ cells into meiosis during HCG‐induced spermatogenesis in the eel.
Association of the sea urchin EGF‐related peptide, EGIP‐D, with fasciclin I‐related ECM proteins from the sea urchin Anthocidaris crassispina
Authors: Hirate Yoshikazu , Tomita Kazuo , Yamamoto Shinji , Kobari Kazuki , Uemura Isao , Yamasu Kyo , Suyemitsu Takashi
Published: March 2003

Exogastrula‐inducing peptides (EGIP) of the sea urchin Anthocidaris crassispina are endogenous peptides related to epidermal growth factor (EGF), which induce exogastrulation in the embryo. Recently, a protein(s) from sea urchin embryos that binds to one of the EGIP, EGIP‐D (EGIP‐D‐binding protein, EBP) was purified. The isolation and characterization of the cDNA clones for two EBP proteins (EBP‐α and EBP‐β) is reported. The two EBP proteins were highly similar in structure to each other; both possessed putative cell‐binding sites and two repeated sequences characteristically seen in the insect neuronal cell adhesion protein, fasciclin I. The EBP showed similarity with other sea urchin proteins HLC‐32, Bep1, and Bep4. It has been confirmed that bacterially expressed EBP proteins associate with EGIP‐D as does native EBP, suggesting the interaction between EGF‐related proteins and fasciclin I‐related proteins. An EBP transcript of 1.4 kb was strongly expressed in immature ovaries but not in immature testes. A somewhat lower level of the transcript existed in unfertilized eggs and the amount gradually declined to an almost undetectable level by the pluteus stage. The EBP proteins were present throughout embryonic development at nearly constant levels. Although most of the proteins were distributed rather evenly in the cytoplasm, a small portion was detected on the apical surface of blastomeres and ectodermal cells, showing that EBP are components of the hyaline layer.
Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos
Authors: Ishii Mamoru , Mitsunaga‐Nakatsubo Keiko , Kitajima Takashi , Kusunoki Shinichiro , Shimada Hiraku , Akasaka Koji
Published: March 2003

In spite of their potential importance in evolution, there is little information about Hox genes in animal groups that are related to ancestors of deuterostome. It has been reported that only two Hox genes (Hbox1 and Hbox7) are expressed significantly in sea urchin embryos. Expression of Hbox1 protein is restricted to the aboral ectoderm, and Hbox7 expression is restricted to oral ectoderm, endoderm and secondary mesenchyme cells in sea urchin embryos after the gastrula stage. With the aim of gaining insight into the role of Hbox1 and Hbox7 in sea urchin development, Hbox1 and Hbox7 overexpression experiments were performed. Overexpression of Hbox1 repressed the development of oral ectoderm, endoderm and mesenchyme cells. On the contrary, overexpression of Hbox7 repressed the development of aboral ectoderm and primary mesenchyme cells. The data suggest that Hbox1 and Hbox7 are expressed in distinct non‐overlapping territories, and overexpression of either one inhibits territory‐specific gene expression in the domain of the other. It is proposed that an important function of both Hbox1 and Hbox7 genes is to maintain specific territorial gene expression by each one, in its domain of expression, while repressing the expression of the other in this same domain.
Expression of murine early embryonic antigens, SSEA‐1 and antigenic determinant of EMA‐1, in embryos and ovarian follicles of a teleost medaka (Oryzias latipes)
Authors: Sasado Takao , Kani Shuichi , Washimi Kazuya , Ozato Kenjiro , Wakamatsu Yuko
Published: March 2003

Stage‐specific embryonic antigen‐1 (SSEA‐1) and the antigenic determinant of monoclonal antibody EMA‐1 are expressed in a stage‐specific manner in mouse early embryos. To study whether these antigens generally exist in fish, expression of the antigens was examined in embryos, ovarian follicles, and adult tissues of a teleost medaka (Oryzias latipes), using immunohistochemical techniques. In 1‐cell‐stage embryos, these carbohydrate antigens were found in numerous cytoplasmic granules in the blastodisc and the cortical cytoplasm. These granules gradually decreased in number as the embryos developed. In 4‐cell‐stage embryos, the antigens appeared on the cleavage planes and were located on the cleavage planes within the blastoderm in the following cleavage stages. In blastula‐stage embryos, the expression was ubiquitously found on the cell surface of blastomeres. At the mid‐gastrula stage, the antigens were restricted to the enveloping layer, yolk syncytial layer, and cortical cytoplasm, but were rarely found in deep cells that contribute to formation of the embryonic body. In later‐stage embryos and adult fish, the antigens were located in various tissues. In ovarian follicles, the antigens were found in granules of oocytes and granulosa cells. These observations were basically consistent with those in mice; however, expression in 1‐cell‐stage embryos and ovarian follicles has not been observed in mice. This unexpected finding suggests that the antigens are produced in granulosa cells and transferred to 1‐cell‐stage embryos via oocytes, and that the antigens involved in the early developmental process are maternally prepared in teleosts.
On the mechanism of ooplasmic segregation in single‐cell zebrafish embryos
Authors: Leung Christina F. , Webb Sarah E. , Miller Andrew L.
Published: March 2003

It has been previously shown that localized elevations of free cytosolic calcium are associated with a morphological contraction in the forming blastodisc and animal hemisphere cortex during ooplasmic segregation in zebrafish zygotes. It was subsequently proposed, in a hypothetical model, that these calcium transients might be linked to the contraction of a cortically located actin microfilament network as a potential driving force for segregation. Here, by labeling single‐cell embryos during the major phase of segregation with rhodamine‐ phalloidin, direct evidence is presented to indicate that the surface contraction was generated by an actin‐based cortical network. Furthermore, while zygotes incubated with colchicine underwent normal ooplasmic segregation, those incubated with cytochalasin B did not generate a constriction band or segregate to form a blastodisc. During segregation at the single‐cell stage, ooplasm simultaneously moved in two directions: toward the blastodisc within the so‐called axial streamers, and toward the vegetal pole in the peripheral ooplasm. The velocities of both axial and peripheral streaming movements are reported. By injection of a fluorescein isothiocyanate (FITC)‐labeled 2000 kDa dextran into the peripheral ooplasm it was demonstrated that a portion of it feeds into the bases of the extending streamers, which helps to explain the lack of accumulation of ooplasm at the vegetal pole. These new data were incorporated into the original model to link the bipolar ooplasmic movements with the calcium‐modulated, actin‐mediated contraction of the animal hemisphere cortex as a means of establishing and driving ooplasmic segregation in zebrafish.
Cloning of a cDNA for Xenopus prolactin receptor and its metamorphic expression profile
Authors: Yamamoto Takashi , Nakayama Yuki , Tajima Tatsuya , Abe Shin‐Ichi , Kawahara Akira
Published: March 2003

A pituitary hormone, prolactin (PRL) shows various effects on cellular metabolism in amphibians, such as stimulation of larval tissue growth and inhibition of metamorphic changes. All these effects are mediated by its cell surface receptor. However, lack of information on PRL receptor (PRL‐R) gene expression has made the physiological importance of the PRL/PRL‐R system obscure in amphibian metamorphosis. Hence, a Xenopus PRL‐R cDNA was cloned, its structure was characterized, and specific binding of PRL to Xenopus PRL‐R expressed in COS‐7 cells was confirmed. In adult tissues, high level expression was found in the lung, heart, brain, thymus and skin, and low level in the oviduct, kidney and spinal cord. The developmental expression pattern showed that PRL‐R messenger ribonucleic acid (mRNA) was expressed in the brain and tail from premetamorphosis and the level increased toward late metamorphosis, suggesting that PRL may inhibit the metamorphic changes in those organs. The level of brain PRL‐R mRNA reached a peak just at the start of the metamorphic climax stages and then decreased, whereas in the tail, mRNA expression peaked at late metamorphosis. In the kidney, mRNA expression increased and reached a maximum level at the end of metamorphosis. The results obtained were discussed in relation to metamorphosis.
Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton
Authors: Kitajima Takashi , Urakami Hiroshi
Published: March 2003

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a ‘spicule blot’ procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c‐axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c‐axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three‐dimensionally branched spicules.
Bone morphogenetic proteins and retinoic acid induce human endogenous retrovirus HERV‐K expression in NT2D1 human embryonal carcinoma cells
Authors: Caricasole Andrea , Oostwaard Dorien Ward‐van , Mummery Christine , Raaij Adriana van den Eijnden‐van
Published: March 2003

Expression of the HERV‐K human endogenous retrovirus is very low in normal and tumor tissue, but is readily detected in testicular germ cell tumors (TGCT). NT2D1 human embryonal carcinoma cells represent in vitro models for the stem cells of TGCT, and can be differentiated by treatment with bone morphogenetic proteins (BMP) or retinoic acid (RA). In a search for BMP target genes in NT2D1 cells, HERV‐K was identified as an early BMP and RA target. It was shown that HERV‐K expression was induced upon treatment of NT2D1 cells with BMP or with RA, but not with activin or transforming growth factor (TGF)‐β. Induction of HERV‐K expression was rapid but transient, with transcripts becoming undetectable in differentiated NT2D1 cultures. Thus NT2D1 cells provide a suitable in vitro system for the study of the factors controlling HERV‐K expression during cellular differentiation, which may play a role in HERV‐K expression in TGCT.
Desensitization of IP3‐induced Ca2+ release by overexpression of a constitutively active Gqα protein converts ventral to dorsal fate in Xenopus early embryos
Authors: Kume Shoen , Saneyoshi Takeo , Mikoshiba Katsuhiko
Published: March 2003

The constitutively active Gqα mutant construct (GqαQ‐L) in Xenopus early embryos was overexpressed and the effects on dorsoventral patterning examined. It was found that prolonged stimulation of inositol 1,4,5‐trisphosphate (IP3)‐Ca2+ signaling by overexpression of GqαQ‐L led to desensitization of IP3‐induced Ca2+ release (IICR). Desensitization of IICR on the ventral side specifically induced an ectopic dorsal axis due to the conversion of ventral marginal mesoderm to adopt a dorsal fate. This effect of desensitization resembles that of inhibitory antibodies against the IP3 receptor, as reported previously. These results strengthen the earlier finding that active IP3‐Ca2+ signaling functions in ventral signaling during the early embryonic development of Xenopus. Furthermore, the nature of downregulation of the Xenopus IP3 receptor through continuous stimulation of IP3‐Ca2+ signaling might play a role in regulating endogenous IP3‐Ca2+ signaling in Xenopus early development.
Homoiogenetic regulation through the ectoderm on localized expression of the hatching gland phenotype in the head area of Xenopus embryos
Authors: Tamori Yoichiro , Mita Koichi , Katagiri Chiaki
Published: March 2003

Ectoderm pieces explanted from embryos of Xenopus laevis were cultured and examined for differentiation of hatching gland cells, using immunoreactivity against anti‐XHE (Xenopus hatching enzyme) as a marker. The anterio‐dorsal ectoderm excised from stage 12–13 (mid–late gastrula) embryos developed hatching gland cells. Meanwhile, the posterio‐, but not the anterio‐dorsal ectoderm from stage 11 (early gastrula) embryos developed these cells, although it is not fated to do so during normogenesis. This hatching gland cell differentiation from stage 11 posterior ectoderm was not affected by conjugated sandwich culture with the mesoderm but was suppressed when explants contained an anterior portion of the ectoderm. Conjugated cultures of anterior and posterior portions of the ectoderm in various combinations indicated that differentiation of hatching gland cells from stage 11 posterior and stage 12 anterior portions was suppressed specifically by stage 11 anterior ectoderm. Northern blot analyses of cultured explants showed that XHE was expressed in association with XA‐1, suggesting its dependence on the anteriorized state. These results indicate that the planar signal(s) emanating from stage 11 anterior ectoderm participates in suppression of the expression of the anteriorized phenotype so that an ordered differentiation along the anteroposterior axis of the surface ectoderm is accomplished.
Pamlin‐induced tyrosine phosphorylation of SUp62 protein in primary mesenchyme cells during early embryogenesis in the sea urchin, Hemicentrotus pulcherrimus
Authors: Katow Hideki , Washio Masahiko
Published: March 2003

Ingression of primary mesenchyme cells (PMC) is associated with the encounter of basal lamina including pamlin. It was found that sea urchin embryos have a protein that binds antihuman focal adhesion kinase (FAK) antibodies, yet it has a 62 kDa homo‐dimeric structure. Thus, this protein was distinctive from known FAK, and was named SUp62. In mesenchyme blastulae, one of the subunits increased its apparent molecular mass slightly but distinctively, then restored the original molecular mass in early gastrulae. This temporal and stage‐specific shifting of the molecular mass was associated with the occurrence of tyrosine phosphorylation of a subunit that did not increase the apparent molecular mass. Herbimycin A induced the hyperphosphorylation of tyrosine residues of SUp62, and inhibited the occurrence of molecular mass shifting. Immunohistochemistry showed a strong positive signal of SUp62 and phosphotyrosine in PMC. Herbimycin A also severely but reversibly inhibited PMC dissociation, migration and gastrulation. Tyrosine phosphorylation of SUp62 was induced when PMC were incubated with pamlin in vitro, and it was initiated within 10 min after onset of the incubation. It reached its peak in 1 h, and declined gradually in the next 1 h, indicating that pamlin‐induced tyrosine phosphorylation of SUp62 occurs closely associated with acquiring PMC migration activity.
Involvement of Frizzled‐10 in Wnt‐7a signaling during chick limb development
Authors: Kawakami Yasuhiko , Wada Naoyuki , Nishimatsu Shin‐ichiro , Nohno Tsutomu
Published: March 2003

The dorsal ectoderm of the limb bud is known to regulate anterior–posterior patterning as well as dorsal– ventral patterning during vertebrate limb morphogenesis. Wnt‐7a, expressed in the dorsal ectoderm, encodes a key molecule implicated in these events. In the present study, chicken frizzled‐10 (Fz‐10) encoding a Wnt receptor was used to study mechanisms of Wnt‐7a signaling during chick limb patterning, because its expression is restricted to the posterior‐distal region of the dorsal limb bud. Fz‐10 transcripts colocalize with Sonic hedgehog (Shh) in the dorsal side of stages 18–23 chick limb buds. It was demonstrated that Fz‐10 interacts with Wnt‐7a to induce synergistically the expression of Wnt‐responsive genes, such as Siamois and Xnr3, in Xenopus animal cap assays. In the chick limb bud, Fz‐10 expression is regulated by Shh and a signal from the dorsal ectoderm, presumably Wnt‐7a, but not by signals from the apical ectodermal ridge. These results suggest that Fz‐10 acts as a receptor for Wnt‐7a and has a positive effect on Shh expression in the chick limb bud.