bluuebunny/crossref_metadata_2025 huggingface dataset:
Progress In Reducing The Burden Of Stroke*
Authors: Chalmers John , Chapman Neil
Published: March 2003

1. The burden of stroke worldwide is growing rapidly, driven by an ageing population and by the rapid rate of urbanization and industrialization in the developing world. There are approximately 5 million fatal and 15 million non‐fatal strokes each year and over 50 million survivors of stroke alive, worldwide, today.

2. The most important determinant of stroke risk is blood pressure, with a strong, continuous relationship between the level of the systolic and diastolic pressures and the risk of initial and recurrent stroke, in both Western and Asian populations.

3. Randomized clinical trials have clearly demonstrated that blood pressure lowering reduces the risk of initial stroke by 35–40% in hypertensive patients; but, until recently, there was no conclusive evidence that blood pressure lowering was effective in the secondary prevention of stroke.

4. The Perindopril Protection Against Recurrent Stroke Study (PROGRESS) has provided definitive evidence that blood pressure lowering in patients with previous stroke or transient ischaemic attack (TIA) reduces the incidence of secondary stroke by 28%, of major vascular events by 26% and of major coronary events by 26%. These reductions were all magnified by approximately 50% in a subgroup of patients in whom the angiotensin‐converting enzyme inhibitor perindopril was routinely combined with the diuretic indapamide.

5. Successful global implementation of a treatment with perindopril and indapamide in patients with a history of stroke or TIA would markedly reduce the burden of stroke and could avert between 0.5 and one million strokes each year, worldwide.
RENIN GENE EXPRESSION: THE SWITCH AND THE FINGERS
Authors: Morris Brian J , Adams David J , Weyden Louise van der
Published: March 2003

1. Now that many of the factors and control elements that regulate renin transcription have been identified, the scene is set to address the question of the mode of control.

2. Based on current gene control theories, either renin gene transcription in each cell undergoes gradual responses over a continuous range or transcription is switched completely on or completely off. The latter model of ‘binary’ or ‘variegated’ expression fits with observations such as the ‘recruitment’ of new cells for renin expression during strong physiological stimulation and the progressive switching off of expression during development.

3. The renin gene offers an excellent general model for testing the mode of control of genes that are subject to continuous modulatory influences from the demands of physiological perturbations. This is because the promoter is well characterized and is subject to the influence of a strong far‐upstream enhancer, one of the key elements of the variegation model.

4. Renin is also controlled at the post‐transcriptional level and this, like transcriptional control, involves cAMP mechanisms. We have cloned the human and mouse homologues of a protein (ZNF265) that is important in renin mRNA processing and stability. This uses ‘zinc fingers’ to bind the mRNA. The role of this and other proteins in splicing and stabilization of mRNA is now being elucidated.

5. Unravelling the mechanisms that determine rate of supply of renin mRNA to the biosynthetic machinery is being assisted by advances in concepts and techniques in the rapidly moving field of genomics.
Glutamatergic Neural Transmission In The Nucleus Tractus Solitarius:N‐Methyl‐D‐Aspartate Receptors
Authors: Bonham Ann C , Chen Chao‐Yin
Published: March 2003

1. The nucleus tractus solitarius (NTS) is the first central site where the reflex control of autonomic, including baroreceptor, reflex function is coordinated. Autonomic signals are transmitted from the first‐order visceral afferent fibres to second‐order NTS neurons byL‐glutamate. It is well established that activation of theα‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole proprionic acid (AMPA) receptors, which mediate the fast component ofL‐glutamate signalling, is required for generating changes in membrane potentials of the second‐order NTS neurons. The contribution of the slower‐developing, longer‐lastingN‐methyl‐D‐aspartate (NMDA) receptor‐mediated component of glutamate signalling to synaptic transmission at these synapses is less well understood.

2. The aim of this work is to highlight evidence that functional NMDA receptors exist on second‐order NTS neurons in autonomic, including baroreceptor, afferent pathways by determining whether NMDA receptors can be activated by: (i) exogenous application of NMDA; and (ii) endogenous release ofL‐glutamate from autonomic afferent fibres. Studies were performed on second‐order neurons in transverse and horizontal brainstem slices containing the intermediate NTS and the tractus solitarius. Second‐order NTS neurons were identified by electrophysiological criteria or by attached fluorescent‐labelled aortic depressor nerve (ADN) boutons.

3. N‐Methyl‐D‐aspartate (50 nmol–2μmol) dose‐dependently evoked excitatory post‐synaptic currents in second‐order NTS neurons (P= 0.004;n= 4). The NMDA receptor‐mediated currents were also synaptically evoked by low‐frequency stimulation of the autonomic afferent fibres in the tractus solitarius. The NMDA receptor‐mediated currents were blocked by the NMDA receptor antagonist AP5 (n= 7;P= 0.027).

4. The findings suggest that functional NMDA receptors exist on second‐order NTS neurons. While the NMDA receptor‐ mediated currents may not be required for signal transmission when the second‐order neurons are at resting membrane potential, their activation may help to modulate autonomic signal transmission in the NTS under conditions in which the membrane is depolarized by high frequency or convergent inputs.
Baicalin‐Induced Vascular Response In Rat Mesenteric Artery: Role Of Endothelial Nitric Oxide
Authors: Huang Yu , Tsang Suk Ying , Yao Xiaoqiang , Lau Chi Wai , Su Ya Lun , Chen Zhen Yu
Published: March 2003

1. Baicalin was isolated and purified from the dry roots of Scutellaria baicalensis Georgi (Huangqin; a traditionally used Chinese medicinal herb) and its effect on the contractility of rat isolated mesenteric arteries was investigated and the role of the endothelium was examined.

2. The concentration‐dependent contractile response to U46619 was enhanced by 10–5 mol/L baicalin in endothelium‐intact rings, but this effect was abolished in the presence of 10–4 mol/L NG‐nitro‐L‐arginine or in endothelium‐denuded rings.

3. Pretreatment of endothelium‐intact rings with baicalin (3 × 10–5 mol/L) markedly attenuated the relaxant response to A23187, thapsigargin and acetylcholine.

4. The present results indicate an important role for endothelial nitric oxide (NO) in the vascular response to baicalin. Baicalin appears to inhibit NO production and release in the endothelium and this mechanism is likely to be responsible for the enhancement of the U46619‐induced contraction and for inhibition of endothelial NO‐mediated relaxation by baicalin in rat mesenteric artery.
Nicorandil Improves Myocardial High‐Energy Phosphates In Postinfarction Porcine Hearts
Authors: Murakami Yo , Wu Xiaoyun , Zhang Jianyi , Ochiai Koichi , Bache Robert J , Shimada Toshio
Published: March 2003

1. Nicorandil is a potent vasodilator combining the effects of a nitrate with an ATP‐sensitive potassium channel (KATP) opener. Because the postinfarct remodelled heart has increased vulnerability to subendocardial hypoperfusion, it is possible that the vasodilator effects of nicorandil could cause transmural redistribution of blood flow away from the subendocardium. Alternatively, the KATP channel opening effects of nicorandil could exert a beneficial effect on mitochondrial respiration. Consequently, the present study was performed to examine the effect of nicorandil on energy metabolism in the postinfarct heart.

2. Studies were performed in swine in which myocardial infarction produced by proximal left circumflex coronary artery ligation had resulted in left ventricular remodeling. [31P] nuclear magnetic resonance spectroscopy (MRS) was used to examine the myocardial energy supply/demand relationship across the left ventricular wall while the transmural distribution of blood flow was examined with radioactive microspheres. Data were obtained during baseline conditions and during infusion of nicorandil (100 μg, i.v., followed an infusion of 25 μg/kg per min).

3. Nicorandil caused coronary vasodilation with a preferential increase in subepicardial flow; however, subendocardial flow also increased significantly. Nicorandil had no significant effect on the rate–pressure product or myocardial oxygen consumption. The ratio of phosphocreatine (PCr)/ATP determined with MRS was abnormally depressed in remodelled hearts (2.01 ± 0.11, 1.85 ± 0.10 and 1.59 ± 0.11 for subepicardium, midwall and subendocardium, respectively) compared with normal (2.22 ± 0.11, 2.01 ± 0.15 and 1.80 ± 0.09, respectively). Nicorandil had no effect on the high‐energy phosphate content of normal hearts. However, nicorandil increased the PCr/ATP ratio in the subendocardium of remodelled hearts from 1.59 ± 0.11 to 1.87 ± 0.10 (P < 0.05).

4. Although nicorandil caused modest redistribution of blood flow away from the subendocardium of the postinfarct left ventricle, this was associated with an increase of the PCr/ATP ratio towards normal. These results suggest that nicorandil exerts a beneficial effect on energy metabolism in the subendocardium of the postinfarct remodelled left ventricle.
Effect Of Grapefruit Juice On Plasma Chloroquine Kinetics In Mice
Authors: Ali BH , Al‐Qarawi A , Mousa HM
Published: March 2003

1. It is known that grapefruit juice (GFJ) may interact with drugs concomitantly administered by inhibiting first‐pass metabolism during the intestinal absorption phase. However, its interaction with chloroquine has not been studied previously.

2. Grapefruit juice (4 mL/kg) was given orally to mice 1 h prior to oral administration of chloroquine (100 mg/kg) and the concentration of the latter drug was measured fluorometrically in the plasma 0, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 18 and 24 h after its administration.

3. The mean (±SEM) of area under the curve values after administration of water ± control) and GFJ were 5.34 ± 0.38 and 7.01 ± 0.66 mg·h/L, respectively. The corresponding mean Cmax values were 763.4 ± 39.1 and 859.2 ± 45.2 mg/L and the corresponding Tmax values (median) were 2.65 and 2.95 h.

4. The results suggest that GFJ coingestion increased the plasma concentration of chloroquine and altered some kinetic parameters of chloroquine. The clinical significance of this interaction in patients with malaria needs to be investigated.
Roles of adenosine in skeletal muscle during systemic hypoxia
Authors: Marshall Janice M
Published: March 2003

1. The present review is concerned with the effects of acute systemic hypoxia on the gross vascular conductance of skeletal muscle (MVC) and on the behaviour of muscle microcirculation.

2. On the basis of experiments performed in the rat, it is argued that adenosine released from the vascular endothelium plays a major role in dilating muscle vasculature by acting on adenosine A1 receptors.

3. The dilatation of the proximal arterioles is primarily important in increasing MVC and in limiting the fall in O2 delivery to muscle. It is suggested that the action of adenosine on proximal arterioles is dependent on nitric oxide (NO) rather than mediated by NO, such that adenosine dilates the proximal arterioles via other mechanisms when synthesis of NO is blocked.

4. In contrast, dilatation of terminal arterioles, particularly in regions within muscle where the hypoxia is most severe, helps to improve the distribution of available O2, allowing muscle O2 consumption to be maintained by increased O2 extraction. It is concluded that the action of adenosine on terminal arterioles is mainly mediated by NO arising from stimulation of endothelial A1 receptors.

5. Therefore, adenosine plays a major role in coordinating the behaviour of muscle vasculature such that the relationship between O2 supply and O2 demand can be optimized even when the O2 content of the arterial blood is greatly reduced.
Choleresis and inhibition of biliary lipid secretion induced by piperacillin in the rat
Authors: González Paquita , Mauriz José Luís , Jiménez Rafael , González‐Gallego Javier , Tuñón María Jesús
Published: March 2003

1. The effects of the administration piperacillin on bile flow and biliary lipid secretion were studied in male Wistar rats.

2. Intravenous injection of piperacillin at doses ranging from 0.3 to 3.0 mmol/kg bodyweight led to an increase in its biliary concentration and excretion rate. Maximal biliary excretion was reached at a dose of 2.0 mmol/kg piperacillin.

3. Excretion of the antibiotic into bile was associated with a marked choleresis. A linear relationship was observed between bile flow and piperacillin excretion, 5.7 µL bile being produced per µmol piperacillin excreted into the bile.

4. Continuous i.v. infusion of piperacillin at 2.0 mmol/100 g per min did not result in significant changes in bile acid or cholesterol secretion, but biliary phospholipid secretion was markedly reduced. The inhibitory effect on phospholipid secretion was also present when biliary lipid output had been previously increased by an infusion of taurocholate (200 nmol/100 g per min). Addition of taurocholate did not reverse the impairment of phospholipid secretion induced by piperacillin.

5. These results indicate that acute administration of piperacillin in the rat induces a marked choleresis by stimulating bile acid‐independent bile flow. The significant impairment in phospholipid secretion suggests a specific effect on intracellular supply and/or translocation across the canalicular membrane.
Intravascular granulocyte aggregates caused by the selectin‐binding carbohydrate fucoidin in pig kidneys
Authors: Dittrich Sven , Lippek Frank , Gratopp Alexander , Grosse‐Siestrup Christian , Lange Peter E , Bührer Christoph
Published: March 2003

1. Renal postischaemic reperfusion injury constitutes a significant problem after kidney transplantation. The polysaccharide fucoidin improves postischaemic function in lamb hearts, presumably by blocking selectin‐mediated leucocyte adhesion.

2. In the present study, eight pairs of ischaemic pig kidneys were reperfused in an ex vivo model with autologous blood with or without fucoidin (100 mg/L).

3. Fucoidin resulted in a significant decrease in renal blood flow (45 ± 5 vs 178 ± 22 mL/min per 100 g; P < 0.001) and increased vascular resistance (3.80 ± 0.07 vs 0.60 ± 0.12 mmHg/mL per min per 100 g; P < 0.001).

4. Histological examination revealed granulocyte emboli in afferent glomerular arteries in five of six fucoidin‐treated kidneys and in one of six controls (Fisher's exact test; P < 0.001).

5. In vitro experiments with human granulocytes showed that large granulocyte aggregates were induced by fucoidin at concentrations similar to those used in reperfused kidneys, whereas slightly lower doses of fucoidin prevented l‐selectin‐dependent homotypic granulocyte adhesion.

6. The formation of embolizing granulocyte aggregates defines a narrow therapeutic range for fucoidin and calls into question its experimental use as an inhibitor of selectin‐mediated leucocyte adhesion.
Fluvastatin remodels resistance arteries in genetically hypertensive rats, even in the absence of any effect on blood pressure
Authors: Ledingham Janet M , Laverty Richard
Published: March 2003

1. The aims of the present study were, first, to determine whether, in the genetically hypertensive (GH) rat, fluvastatin would lower blood pressure and remodel mesenteric resistance arteries (MRA) and the basilar artery and, second, to see whether treatment with a combination of fluvastatin and the angiotensin receptor antagonist valsartan would have any extra beneficial effect on blood pressure and vascular remodelling.

2. Male GH rats had tail‐cuff systolic blood pressure (SBP) monitored weekly from the age of 7 to 12 weeks. Groups (n = 12–14) were treated with fluvastatin (4 mg/kg per day), valsartan (5 mg/kg per day), both mixed in with chow, or a combination of fluvastatin 4 mg/kg per day + valsartan 5 mg/kg per day. Untreated GH and a group of normotensive Wistar (N) rats served as control groups.

3. At 12 weeks of age, intra‐arterial (i.a.) blood pressure was measured by femoral cannulation and rats were then perfused (at the SBP of the animal) with Tyrode's solution containing heparin and papaverine followed by 2.5% glutaraldehyde in Tyrode's solution; MRA and basilar arteries were embedded in Technovit. Serial sections were cut and Giemsa stained and stereological methods used to obtain media width, lumen diameter, medial cross‐sectional area (CSA) and the ratio of media width to lumen diameter. Hearts were weighed to determine left ventricular (LV) mass.

4. Fluvastatin had no effect on blood pressure or LV mass, whereas valsartan given alone or with fluvastatin significantly reduced both parameters.

5. In MRA, fluvastatin reduced medial CSA, increased lumen size and, therefore, probably decreased vascular resistance. The media/lumen ratio was reduced to a level below that seen with the combination treatment and to below that of the N group.

6. In the basilar artery, fluvastatin and valsartan showed similar outward remodelling of the lumen and reduction in the media/lumen ratio. The combination treatment group showed, in addition, a reduction in medial CSA and an even lower ratio than the GH group on fluvastatin or valsartan alone or the N group.

7. Although fluvastatin has no effect on blood pressure, it does cause significant remodelling of MRA and the basilar artery. These beneficial structural changes in a peripheral resistance artery bed and in an artery involved in regulating resistance in the brain are worthy of further study.
Nitric oxide inhibits renal cytochrome P450‐dependent epoxygenases in the rat
Authors: Oyekan Adebayo
Published: March 2003

1. Nitric oxide (NO), or peroxynitrite, is known to inhibit haemoproteins, including cytochrome P450 mono‐oxygenases. The present study explores the functional correlates of the inhibition by NO of renal epoxygenase on the vascular responses to arachidonic acid (AA) in the perfused kidney.

2. Control kidneys produce measurable amounts of epoxyeicosatrienoic acids (epoxides), which were increased from 0.6 ± 0.2 to 1.8 ± 0.9 ng/min (P < 0.05) following the addition of AA 5 µg. Sodium nitroprusside (SNP; 100 µmol/L), an NO donor, blunted the basal and AA‐stimulated efflux of epoxides.

3. Sodium nitroprusside at 10 and 100 µmol/L inhibited renal microsomal conversion of [14C]‐AA to epoxides and its hydration products dihydroxyeicosatrienoic acid (diols). Microsomes harvested from rats 3 h after treatment with Escherichia coli endotoxin (lipopolysaccharide; LPS) also inhibited renal epoxygenase activity (81 ± 8%; P < 0.05).

4. In the phenylephrine‐preconstricted and indomethacin (2.8 µmol/L)‐treated kidney, AA at 5, 10 and 25 µg elicited vasodilation that was blunted by miconazole (2 µmol/L), 80 mmol/L KCl, tetraethylammonium (10 mmol/L), a K+ channel blocker, or SNP (100 µmol/L).

5. Vasodilation induced by AA, but not 5,6‐epoxide, was reduced in rats treated with LPS, an effect that was abolished by Nω‐nitro‐l‐arginine (100 mg/kg in drinking water for 10 days).

6. These data suggest that NO inhibits renal epoxygenase activity and inhibits epoxide‐mediated AA‐induced vasodilation in the rat kidney.
Membrane‐bound and releasable nucleotidase activities: Differences in canine mesenteric artery and vein
Authors: Bobalova Janette , Mutafova‐Yambolieva Violeta N
Published: March 2003

1. At least two enzymatic activities are proposed to degrade the extracellular ATP: (i) ubiquitously expressed membrane‐bound enzymes (ecto‐nucleotidases); and (ii) soluble (releasable) nucleotidases that are released during stimulation of sympathetic nerves and break down neuronal ATP. No quantitative data have placed the magnitude of these nucleotidase activities into a physiological perspective of neurovascular control.

2. We studied comparatively the membrane‐bound and releasable nucleotidase activities in canine isolated inferior mesenteric arteries and veins using 1,N6‐etheno(ε)‐nucleotides (i.e. ε‐ATP, ε‐ADP, ε‐AMP and ε‐adenosine) as exogenous substrates. The enzymatic activities were estimated by measuring the disappearance of the ε‐substrate and appearance of ε‐products by means of HPLC–fluorescence detection during either stimulation of sympathetic perivascular nerves (releasable activity) or in the absence of nerve stimulation (ecto‐nucleotidase activity).

3. Incubation of vascular segments with 50 nmol/L ε‐ATP for 60 min resulted in a decrease of the ε‐ATP substrate by 63.5 ± 4.6 and 91.2 ± 6.2% in the artery and vein, respectively. In contrast, the decrease of the ε‐ATP during electrical field stimulation (EFS; 16 Hz, 0.3 msec, 2 min) was 39.8 ± 4.2% in the artery and 13.1 ± 7.3% in the vein. Therefore, the mesenteric arteries demonstrate a greater releasable ATPase activity and a weaker ecto‐ATPase activity than mesenteric veins.

4. The degradation of ε‐ADP and ε‐AMP was similar in both blood vessels under either experimental protocol. The ε‐adenosine was not significantly degraded in the absence or presence of EFS.

5. These data implicate a differential removal of extracellular ATP as a potential mechanism of serving resistance and capacitance in the splanchnic circulation.
Identification of mRNA‐binding proteins during development: Characterization of Bufo arenarum cellular nucleic acid binding protein
Authors: Calcaterra Nora B. , Palatnik Javier F. , Bustos Diego M. , Arranz Silvia E. , Cabada Marcelo O.
Published: March 2003

Ultraviolet irradiation was used to covalently cross‐link poly(A)+RNA and associated proteins in eggs and embryos of the toad Bufo arenarum. Four major proteins with apparent sizes of 60, 57, 45 and 30–24 kDa were identified. It was observed that the same mRNA‐binding proteins were isolated from eggs to gastrula and neural stages of development. The 30 kDa polypeptide, p30, appeared as the main ultraviolet (UV) cross‐linked protein in the developmental stages analyzed. By means of polyclonal antibodies, it was determined that this polypeptide has a cytoplasmic localization and it was detected in liver, eggs and embryos. The presence of p30 was also analyzed by western blot during oogenesis and development. The 30 kDa polypeptide was present in all stages analyzed but it could not be detected in stages I–II of oogenesis. At the neural stage, the relative amount of p30 began to decrease, reaching its lowest levels after stages 26–30 (tail‐bud in Bufo arenarum). On the basis of purification, immunoprecipitation and western blot assays the 30 kDa protein was identified as the Bufo arenarum cellular nucleic acid binding protein.
Retinoic acid given at late embryonic stage depresses sonic hedgehog and Hoxd‐4 expression in the pharyngeal area and induces skeletal malformation in flounder (Paralichthys olivaceus) embryos
Authors: Suzuki Tohru , Oohara Ichiro , Kurokawa Tadahide
Published: March 2003

During the development of pharyngeal cartilages, signal molecules, including sonic hedgehog (shh) and various growth factors, as well as Hox genes are expressed in the pharyngeal area. To elucidate whether shh and Hoxd‐4 function in pharyngeal cartilage formation in teleost jaw and gill primordia, spatial and temporal patterns of shh expression in flounder (Paralichthys olivaceus) embryonic pharynx were examined. The effects of retinoic acid (RA) on shh and Hoxd‐4 expression and the patterning of pharyngeal cartilages were analyzed. At the prim‐5 stage, when cartilage precursor cells aggregate in the pharyngeal primordia, pharyngeal endoderm expressed shh in two domains, in portions of the mandibular and hyoid primordia and in the gill primordia. After a further 40 h, shh domains expanded at the posterior edge of the endoderm of each mandibular, hyoid and gill primordium, concurrent with the growth of the primordia. A new shh expression domain appeared at the endodermal border of the mouth. Retinoic acid treatment depressed shh and Hoxd‐4 expression, and also reduced the amount of expansion of the shh expression domains. Pharyngeal cartilages that formed in these embryos were malformed; their growth direction was shifted posteriorly and size was reduced. This provides the possibility that shh and Hoxd‐4 regulate the growth and direction of pharyngeal cartilage precursor cells and that RA disturbs their expression, causing skeletal malformation.
Expression of Eph receptor tyrosine kinases and their ligands in chick embryonic motor neurons and hindlimb muscles
Authors: Iwamasa Hiroko , Ohta Kunimasa , Yamada Tomoko , Ushijima Kazuo , Terasaki Hidenori , Tanaka Hideaki
Published: March 2003

Evidence is accumulating that Eph receptor tyrosine kinases and their ligands regulate cell migration and axonal guidance during development. It was previously found that one of the Eph receptors, EphA4, is transiently expressed in subsets of chick embryonic motor neurons. Here, the expression of EphA and ephrin‐A subfamily members was further examined, and the dynamic patterns of expression in chick embryonic motor neurons found. EphA3, EphA4, ephrin‐A2, and ephrin‐A5 were also expressed in the connective tissues of limb muscles and EphA3 and EphA4 expressing motor neurons innervated EphA3 and EphA4 expressing limb muscles, respectively. These spatiotemporal expression patterns suggest that EphA and ephrin‐A proteins play important roles in muscle patterning and motor axonal guidance.
A diffusible factor involved in MAP‐kinase ERK2‐regulated development of Dictyostelium
Authors: Maeda Mineko , Kuwayama Hidekazu
Published: March 2003

Mitogen‐activated protein (MAP)‐kinase extracellular signal regulated kinase (ERK2) is essential for regulation of the intracellular cyclic adenosine monophosphate (cAMP) level in Dictyostelium. The mutant lacking ERK2, erk2‐null, is arrested at the pre‐aggregation stage, but develops into a fruiting body in a mixed population of wild‐type and mutant cells. This fact implies that wild‐type cells provide a certain factor that is missing in erk2‐null. It was clarified that both wild‐type strains KAx3 and Ax2 secreted a diffusible factor that enables erk2‐null to develop. The fruiting body formed from erk2‐null cells was smaller than that formed by the wild‐type cells and consisted of a small sorus supported by a slender stalk with a single row of vacuolated stalk cells. The resulting spores were able to germinate and multiply on a bacterial lawn, but they were unable to develop unless the factor was provided. After 8 h of starvation, wild‐type cells started to secrete the factor, which had a molecular mass of less than 3 kDa and was heat stable. The effect of this factor could not be mimicked by either cAMP or folate. Adenylyl cyclase A and cell surface cAMP receptors cAR1 and cAR3 were all indispensable components for the factor to function. Considering the molecular mass and the mode of action, this factor could be a novel one. Possible targets of this factor are discussed in terms of cAMP‐dependent protein kinase activation.
A Drosophila Winged‐helix nude (Whn)‐like transcription factor with essential functions throughout development
Authors: Sugimura Isamu , Adachi‐Yamada Takashi , Nishi Yoshimi , Nishida Yasuyoshi
Published: March 2003

A Drosophila gene, Dwhn (Drosophila whn‐like), encoding a putative transcriptional regulator with a DNA binding domain similar to that of mouse Winged‐helix nude (Whn) was cloned. Analyses of the phenotypes produced by a hypomorphic mutation and transgene expression suggested a role in cell fate decision during the differentiation of the compound eye, wing veins and bristles. During embryonic development, Dwhn expression started ubiquitously followed by more restricted expression in striking contrast to the expression patterns of other Drosophila forkhead (fkh) family genes whose local expression correlate well to their roles as local homeotic genes. This broad expression may correspond to the multiple defects in embryos homozygous for strong alleles, such as defects in the formation of central and peripheral nervous systems, germ band retraction, head involution, and dorsal closure. The DNA binding specificity of Dwhn differed from that of Whn despite the strong sequence conservation in the DNA binding domain. Dwhn is the first invertebrate Whn‐like transcriptional regulator, and should provide insights into the basic functions and evolution of the whn family genes.
Induction of proopiomelanocortin mRNA expression in animal caps of Xenopus laevis embryos
Authors: Holling Tjadine M. , Herp François van , Martens Gerard J. M.
Published: March 2003

To convert animal pole cells of a frog embryo from an ectodermal fate into a neural one, inductive signals are necessary. The alkalizing agent NH4Cl induces the expression of several anterior brain markers and the early pituitary marker XANF‐2 in Xenopus animal caps. Here it is demonstrated that NH4Cl also induced proopiomelanocortin (POMC)‐expressing cells (the first fully differentiated pituitary cell type) in stage 9 and 10 Xenopus animal caps, and that all‐trans retinoic acid, a posteriorizing agent, was able to block this induction when it was administered within 2 h after the start of NH4Cl incubation. Thus, after 2 h, the fate of Xenopus animal cap cells was determined. Microinjection of ribonucleic acid (RNA) encoding noggin, an endogenous neural inducer, led to the induction of POMC gene expression in animal caps of stage 10 embryos, suggesting that noggin represents a candidate mesodermal signal leading to the POMC messenger (m) RNA producing cell type in uncommitted ectoderm. Hence, an alkalizing agent and a neural inducer can generate a fully differentiated POMC cell lineage from Xenopus animal caps.
Cloning and characterization of cDNA for syndecan core protein in sea urchin embryos
Authors: Tomita Kazuo , Yamasu Kyo , Suyemitsu Takashi
Published: March 2003

The cDNA for the core protein of the heparan sulfate proteoglycan, syndecan, of embryos of the sea urchin Anthocidaris crassispina was cloned and characterized. Reverse transcription–polymerase chain reaction (RT‐PCR) was used with total ribonucleic acid (RNA) from late gastrula stage embryos and degenerate primers for conserved regions of the core protein, to obtain a 0.1 kb PCR product. A late gastrula stage cDNA library was then screened using the PCR product as a probe. The clones obtained contained an open reading frame of 219 amino acid residues. The predicted product was 41.6% identical to mouse syndecan‐1 in the region spanning the cytoplasmic and transmembrane domains. Northern analysis showed that the transcripts were present in unfertilized eggs and maximum expression was detected at the early gastrula stage. Syndecan mRNA was localized around the nuclei at the early cleavage stage, but was then found in the ectodermal cells of the gastrula embryos. Western blotting analysis using the antibody against the recombinant syndecan showed that the proteoglycan was present at a constant level from the unfertilized egg stage through to the pluteus larval stage. Immunostaining revealed that the protein was expressed on apical and basal surfaces of the epithelial wall in blastulae and gastrulae.
Hydrogen peroxide induces Src family tyrosine kinase‐dependent activation of Xenopus eggs
Authors: Sato Ken‐ichi , Ogawa Keiko , Tokmakov Alexander A. , Iwasaki Tetsushi , Fukami Yasuo
Published: March 2003

Fertilization is accompanied by a rapid and transient calcium release in eggs, which is required for the onset of zygotic developmental program or ‘egg activation’. Recently, it was found that Src family tyrosine kinase (SFK)‐dependent phospholipase C (PLC) activity is necessary for the calcium transience in fertilized Xenopus eggs. The present study demonstrates that hydrogen peroxide (H2O2) stimulates protein‐tyrosine phosphorylation in Xenopus eggs, which occurs primarily in the egg cortex of the animal hemisphere as revealed by indirect immunofluorescence study. Egg SFK was found to be upregulated by H2O2 while the SFK‐specific inhibitor PP1 effectively blocked H2O2‐induced tyrosine phosphorylation. As in fertilized eggs, PLCγ, but not Shc, was tyrosine‐phosphorylated in H2O2‐treated eggs. H2O2 also caused inositol 1,4,5‐trisphosphate (IP3) production and sustained calcium release. After limited application of H2O2, elevated SFK activity and tyrosine phosphorylation were quickly reversed. Under such conditions, eggs showed cortical contraction and dephosphorylation of p42 MAP kinase, both of which are indicative of egg activation. These egg activation events, as well as H2O2‐induced IP3 production and calcium release, were sensitive to PP1 and PLC inhibitor U‐73122. Together, the present study demonstrated that H2O2 can mimic, at least in part, early events of Xenopus egg activation that require an SFK‐dependent PLC pathway.
Evidence for multiple sequences and factors involved in c‐myc RNA stability during amphibian oogenesis
Authors: Lefresne J. , Lemaitre J. M. , Selo M. , Goussard J. , Mouton C. , Andeol Y.
Published: March 2003

To investigate the molecular mechanisms regulating c‐myc RNA stability during late amphibian oogenesis, a heterologous system was used in which synthetic Xenopus laevis c‐myc transcripts, progressively deleted from their 3′ end, were injected into the cytoplasm of two different host axolotl (Ambystoma mexicanum) cells: stage VI oocytes and progesterone‐matured oocytes (unfertilized eggs; UFE). This in vivo strategy allowed the behavior of the exogenous c‐myc transcripts to be followed and different regions involved in the stability of each intermediate deleted molecule to be identified. Interestingly, these specific regions differ in the two cellular contexts. In oocytes, two stabilizing regions are located in the 3′ untranslated region (UTR) and two in the coding sequence (exons II and III) of the RNA. In UFE, the stabilizing regions correspond to the first part of the 3′ UTR and to the first part of exon II. However, in UFE, the majority of synthetic transcripts are degraded. This degradation is a consequence of nuclear factors delivered after germinal vesicle breakdown and specifically acting on targeted regions of the RNA. To test the direct implication of these nuclear factors in c‐myc RNA degradation, an in vitro system was set up using axolotl germinal vesicle extracts that mimic the in vivo results and confirm the existence of specific destabilizing factors. In vitro analysis revealed that two populations of nuclear molecules are implicated: one of 4.4–5S (50–65 kDa) and the second of 5.4–6S (90–110 kDa). These degrading nuclear factors act preferentially on the coding region of the c‐myc RNA and appear to be conserved between axolotl and Xenopus. Thus, this experimental approach has allowed the identification of specific stabilizing sequences in c‐myc RNA and the temporal identification of the different factors (cytoplasmic and/or nuclear) involved in post‐transcriptional regulation of this RNA during oogenesis.
Early expressed genes showing a dichotomous developing pattern in the lancelet embryo
Authors: Yasui K. , Saiga H. , Wang Y. , Zhang P. J. , Semba I.
Published: March 2003

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate‐like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior‐posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp‐related gene, Bbbmp2/4, and two wnt‐related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf‐3β class genes. The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region, respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid‐dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling. The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior‐specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.
Two members of the IgLON family are expressed in a restricted region of the developing chick brain and neural crest
Authors: Kimura Yoshishige , Katoh Aki , Kaneko Tomomi , Takahama Kazuo , Tanaka Hideaki
Published: March 2003

The precise expression patterns of two IgLON genes, CEPU‐1 and limbic system‐associated membrane protein (LAMP), were studied during early embryogenesis. It was found that expression of both was localized to restricted regions of the brain and neural crest. In the developing neural tube, CEPU‐1 was expressed in the isthmus and a restricted region of the hindbrain, whereas LAMP was expressed in the anterior midbrain. Most neural crest cells expressed LAMP, whereas CEPU‐1 expression was limited to crest cells derived from the hindbrain. These results suggest that members of the IgLON family have important roles during embryogenesis, particularly in brain formation and differentiation.
Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration
Authors: Hayashi Toshinori , Yamagishi Atsushi , Kuroiwa Atsushi , Mizuno Nobuhiko , Kondoh Hisato , Okamoto Mitsumasa
Published: March 2003

The analysis of newt lens regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of lens regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into lens cells in newt lens regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene‐transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in lens regeneration, Pax6, which is suggested to be involved in normal eye development and lens regeneration, was transfected. Ectopic expression of lens‐specific crystallins was obtained in cells that show no such activity in normal lens regeneration. These results made it possible for the first time to analyze the molecular mechanism of lens regeneration in the adult newt.
Developmental study of the different effects on the hybrid sterility of KitW and KitW–v alleles paired with KitS from Mus spretus
Authors: El‐Shazly Samir , Okano Shinya , Asano Atsushi , Watanabe Tomomasa
Published: March 2003

The combination of the KitW or KitW–n mutant alleles and KitS from Mus spretus results in male hybrid sterility with small testes. In the present study, reproduction of the combination between KitW–v and KitS alleles was examined. The KitW–v/KitS male was fertile and the histologic structure was normal; the seminiferous tubules showed all of the normal stages of spermatogenesis. The postnatal development of the testis at 8, 12, 16 and 20 days was also studied in the fertile +Kit/+Kit and KitW–v/KitS males and the sterile KitW/KitS. The results showed that at 8 days there was no noticeable difference among the three genotype combinations, while from 12 to 20 days spermatogenesis in the KitW/KitS male nearly stopped before the meiosis stage. The expression of Kit receptor protein from the KitS allele in the sterile testis of the KitW/KitS male was confirmed using western blot analysis. The Kit ligand derived from M. spretus showed two amino acid changes in the extracellular domain compared with that from C57BL and it appears that the ligand–receptor interaction between C57BL and SPR may influence the male hybrid sterility of KitW/KitS.
Ascidian Wnt‐5 gene is involved in the morphogenetic movement of notochord cells
Authors: Sasakura Yasunori , Makabe Kazuhiro W.
Published: March 2003

Wnt proteins play important roles in many developmental events. Wnts are divided into two groups according to biological function. The Wnt‐5a class proteins function in morphogenetic movement during embryogenesis. Previously, a Wnt‐5 homolog has been isolated from the ascidian, Halocynthia roretzi. HrWnt‐5 is expressed in the notochord until the tail‐bud stage, implying a role in the notochord. In this study, the function of HrWnt‐5 was investigated. When HrWnt‐5 mRNA was injected into fertilized eggs, the embryos showed morphologic defects at around the neurula stage. The anterior–posterior axis was shorter than in control embryos. These defects were caused by the abnormal movement of notochord cells. However, the overexpression of HrWnt‐5 mRNA did not affect the differentiation of tissues, suggesting that HrWnt‐5 solely regulates the morphogenetic movement. Although endogenous HrWnt‐5 is expressed in the notochord, the overexpression of HrWnt‐5 mRNA caused the defects, suggesting that the amount of HrWnt‐5 mRNA in the notochord is strictly regulated. These results suggest that HrWnt‐5 regulates the morphogenetic movement of notochord cells during ascidian embryogenesis.
Cloning and characterization of the T‐box gene Tbx6 in Xenopus laevis
Authors: Uchiyama Hideho , Kobayashi Teruaki , Yamashita Akio , Ohno Shigeo , Yabe Shigeharu
Published: March 2003

Tbx6 is a member of the T‐box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6‐expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.
Serosa membrane plays a key role in transferring vitellin polypeptides to the perivitelline fluid in insect embryos
Authors: Fausto Anna Maria , Gambellini Gabriella , Mazzini Massimo , Cecchettini Antonella , Locci Maria Teresa , Masetti Massimo , Giorgi Franco
Published: March 2003

In mid‐embryogenesis, the stick insect Carausius morosus comes to be comprised of three distinct districts: the embryo proper, the yolk sac and the perivitelline fluid. A monolayered epithelium, the so‐called serosa membrane, encloses the yolk sac and its content of vitellophages and large yolk granules. During embryonic development, the yolk sac declines gradually in protein concentration due to Vt polypeptides undergoing limited proteolysis to yield a number of Vt cleavage products of lower molecular weights. mAbs 1D1 and 5H11 are monoclonal antibodies raised against some of the Vt cleavage products generated by this process in the yolk sac. At the confocal microscope, antibody fluorescence is initially associated with a few yolk granules, while it is gradually displaced in the cytosolic spaces of the vitellophages. With the proceeding of embryonic development, label appears also in the serosa membrane in the form of clustered dots. At the ultrastructural level, gold particles are initially associated with the vitellophages that are labeled on a few yolk granules and in the cytosolic space flanking the yolk granules. Subsequently, the serosa cells become labeled on vesicles close to the yolk granules or just underneath the plasma membrane. Inside the serosa cells, label is also associated with granules budding from the Golgi apparatus, but never with the intercellular channels percolating the serosa membrane. These observations are interpreted as indicating that Vt cleavage products leak out from the yolk granules into the cytosolic spaces of the vitellophages and are eventually transferred to the perivitelline fluid via transcytosis through the serosa cells.
Role of syndecan in the elongation of postoral arms in sea urchin larvae
Authors: Tomita Kazuo , Yamasu Kyo , Suyemitsu Takashi
Published: March 2003

Ac‐SYN is the core protein of a cell surface proteoglycan of the sea urchin Anthocidaris crassispina. To examine the functions of Ac‐SYN, embryos were cultured in the presence of affinity‐purified antibody against Ac‐SYN. At the late pluteus stage, severe inhibition of elongation of the postoral arms was seen in treated embryos compared with control embryos. Blastocoeleic microinjection of the antibody did not affect morphogenesis. The relationship between the number of cells in the postoral arms and the length of the postoral rods was investigated in normal embryos. This showed that postoral arm elongation has two phases: the first phase accompanies the increase in cell numbers while the second does not. The syndecan antibody inhibited the increase in cell numbers in the postoral arms. Furthermore, in the treated embryos, cell numbers continued to increase normally until 31 h post fertilization (hpf), while cell division stopped after 31 hpf. These results suggest that Ac‐SYN participates in postoral arm formation via cell division in sea urchin embryos.
Possible role of the 38 kDa protein, lacking in the gastrula‐arrested Xenopus mutant, in gastrulation
Authors: Tanaka Tetsuya S. , Ikenishi Kohji
Published: March 2003

An acidic, 38 kDa protein that is present in Xenopus wild‐type embryos has been previously shown to be lacking in gastrula‐arrested mutant embryos. To gain understanding of the role of this protein, its spatio‐temporal distribution and involvement in gastrulation was investigated using the monoclonal antibody (9D10) against it. The protein was prominent in the cortical cytoplasm of cells facing the outside in the animal hemisphere of embryos until the gastrula stage, and in ciliated epithelial cells of embryos at stages later than the late neurula. When the 9D10 antibody was injected into fertilized wild‐type eggs, they cleaved normally, but most of them had arrested development, always at the early stage of gastrulation, as in the mutant embryos. In contrast, the majority of the control antibody‐injected eggs gastrulated normally and developed further. Cytoskeletal F‐actin, which was mainly observed in the area beneath the plasma membrane facing the outside of the epithelial layer of not only the dorsal involuting marginal zone but also the dorsal, vegetal cell mass of the control antibody‐injected embryos at the early gastrula stage, was scarcely recognized in the corresponding area of the 9D10 antibody‐injected embryos. It is likely that the paucity of the F‐actin caused by the 9D10 antibody inhibition of the 38 kDa protein might lead to a failure of cell movement in gastrulation, resulting in developmental arrest.