bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect Of Kad‐1229, A Novel Hypoglycaemic Agent, On Plasma Glucose Levels After Meal Load In Type 2 Diabetic Rats
Authors: Ichikawa Kiyoshi , Yamato Tokuhisa , Ojima Kazuma , Tsuji Atsutoshi , Ishikawa Kohtaro , Kusama Hiroshi , Kojima Masami
Published: March 2003

1. The effects of KAD‐1229 (a novel non‐sulphonylurea agent), voglibose (an α‐glucosidase inhibitor) and nateglinide (a non‐sulphonylurea antihyperglycaemic agent) on hyperglycaemia induced by a meal load were assessed in diabetic rats.

2. KAD‐1229 suppressed the increase in plasma glucose levels seen after a meal load and the area under the curve for plasma glucose levels (AUCglucose) up to 5 h after the meal load.

3. Voglibose also suppressed the increase in plasma glucose levels; however, a significant decrease in AUCglucose following voglibose was not observed.

4. Nateglinide suppressed the increase in plasma glucose levels at 30 min and 1 h after the meal load; however, plasma glucose levels was above control thereafter and the AUCglucose was not decreased.

5. The results indicate that KAD‐1229 has an antihyperglycaemic effect and KAD‐1229 is suggested to be a suitable agent for controlling post‐prandial hyperglycaemia.
Critical Role Of Gap Junctions In Endothelium‐Dependent Hyperpolarization In Rat Mesenteric Arteries
Authors: Goto Kenichi , Fujii Koji , Kansui Yasuo , Abe Isao , Iida Mitsuo
Published: March 2003

1. Acetylcholine (ACh) evokes endothelium‐dependent hyperpolarization in arterial cells, presumably through endothelium‐derived hyperpolarizing factor (EDHF). The identity of EDHF is still elusive; however, several recent studies suggest the possible involvement of myoendothelial gap junctions in the EDHF response.

2. To elucidate the role of gap junctions in endothelium‐dependent hyperpolarization, we examined the effects of the gap junction inhibitors 18α‐glycyrrhetinic
acid (18α‐GA; 10–4 mol/L) and carbenoxolone (3 ×
10–4 mol/L), a water‐soluble form of 18β‐GA, on hyperpolarization
and relaxation to ACh in rat proximal and distal mesenteric arteries. Experiments
were performed in the presence of indomethacin (10–5 mol/L) and
NG‐nitro‐L‐arginine (10–4 mol/L).

3. In both proximal and distal mesenteric arteries, ACh‐induced hyperpolarization and relaxation were partially inhibited by 18α‐GA and abolished by carbenoxolone.

4. Endothelium‐independent hyperpolarization to levcromakalim, an ATP‐sensitive K+ channel opener, were unaffected by 18α‐GA or carbenoxolone in both arteries.

5. Relaxations to levcromakalim were unaffected by 18α‐GA, but were inhibited somewhat by carbenoxolone in proximal mesenteric arteries.

6. These findings suggest that myoendothelial gap junctions play a critical role in EDHF‐mediated responses in both proximal and distal mesenteric arteries of the rat.
Angiotensin‐converting enzyme inhibition in adult hypertensive rats: A stereological study of renal filtration surface area
Authors: Dunstan HJ , Briscoe TA , Bertram JF , Johnston CI , Black MJ
Published: March 2003

1. Angiotensin‐converting enzyme (ACE) inhibitor treatment leads to beneficial effects on kidney function. The aim of the present study was to determine whether ACE inhibition at high or low doses affects glomerular capillary surface area and length, glomerular number or total renal filtration surface area in rats with established hypertension and, if so, to determine whether these effects are mediated through bradykinin potentiation.

2. Spontaneously hypertensive rats (SHR) were treated with the ACE inhibitor perindopril at either 3 or 0.1 mg/kg per day (high and low doses, respectively) from 16 to 24 weeks of age. Some rats were concomitantly treated with the bradykinin B2 receptor antagonist S16118 (10 nmol/kg per day). Blood pressure was measured twice weekly during the treatment period. At 24 weeks of age, rats were perfusion fixed at 140 mmHg, the kidneys removed, embedded in resin and examined stereologically to estimate glomerular number and volume, length and surface area of glomerular capillaries and total renal filtration surface area.

3. High‐ and low‐perindopril treatment significantly reduced systolic blood pressure compared with control SHR. However, the rats treated with low‐dose perindopril were still considered hypertensive. Neither low‐dose nor high‐dose perindopril treatment had any observable effect on glomerular number (23 876 ± 1201 vs 26 240 ± 1465 glomeruli/kidney, respectively) or volume (2.25 ± 0.21 and 1.96 ± 0.06 × 10−3 mm3, respectively) compared with controls (glomerular number 25 866 ± 1210 glomeruli/kidney; glomerular volume 2.24 ± 0.21 × 10−3 mm3). As a result, there was no significant difference in total renal filtration surface area between any of the experimental groups (8161.6 ± 550.9, 8699.7 ± 427.6, 9081.9 ± 453.6, 8830.2 ± 521.2 and 8559.4 ± 341.4 mm2 for SHR, SHR low‐dose perindopril, SHR low‐dose perindopril + B2 antagonist, SHR high‐dose perindopril and SHR high‐dose perindopril + B2 antagonist, respectively). Coadministration of the bradykinin antagonist had no observable effect on any of the parameters studied.

4. In conclusion, because neither high‐dose nor low‐dose perindopril had any effect on total renal filtration surface area, the observed beneficial effects of ACE inhibition on kidney function are not the result of enhancement in glomerular capillary surface area.
Modulatory effect of Coccinia indica on aortic collagen in streptozotocin‐induced diabetic rats
Authors: Venkateswaran Subramanian , Pari Leelavinothan , Suguna Lonchin , Chandrakasan Gowri
Published: March 2003

1. The effects of Coccinia indica, an indigenous plant used in Ayurvedic medicine in India, on aortic collagen content and its characteristics were assessed in streptozotocin (STZ) diabetic rats.

2. Rats were made diabetic with a single intraperitoneal injection of STZ (45 mg/kg). Blood glucose, hydroxyproline, collagen, extent of glycation, collagen‐linked fluorescence, soluble pattern of pepsin‐soluble collagen, shrinkage temperature, α/β ratio of type I collagen and type I/type III collagen ratio were determined in rats treated with C. indica leaf extract (CLEt; 200 mg/kg for 45 days using an oral intragastric tube).

3. In diabetic rats, the collagen content, as well as the degree of cross‐linking, was increased, as evidenced by increased shrinkage temperature and decreased pepsin solubility. The α/β ratio of type I collagen and the type I/type III collagen ratio of pepsin‐soluble collagen were significantly decreased in STZ diabetic rats.

4. In conclusion, administration of CLEt for 45 days to STZ diabetic rats significantly reduced the accumulation and cross‐linking of collagen. The effects of C. indica (collagen content 23.87 ± 1.52 mg/100 mg tissue (t value = 6.80), extent of cross‐linking 0.893 ± 0.072 mg hydroxyproline/100 mg tissue (t value = 9.0)) were comparable with those of glibenclamide (collagen content 26.18 ± 1.65 mg/100 mg tissue (t value = 4.58), extent of cross‐linking 0.787 ± 0.057 mg hydroxyproline/100 mg tissue (t value = 7.1)), a reference drug.
Outgrowth of pseudopodial cables induced by all‐trans retinoic acid in micromere‐derived cells isolated from sea urchin embryos
Authors: Kuno Shin‐ichi , Kawamoto Manabu , Okuyama Masahiro , Yasumasu Ikuo
Published: March 2003

Cultured cells derived from micromeres of sea urchin embryos underwent pseudopodial cable growth without spicule rod formation in the presence of all‐trans retinoic acid (tRA) or insulin. Pseudopodial cable growth caused by tRA or insulin was inhibited by genistein, a protein tyrosine kinase inhibitor. Phosphorylation of protein tyrosine residue was augmented in the cells treated with tRA or insulin and was inhibited by genistein. Probably, protein tyrosine kinase takes an indispensable part in signal transduction systems for tRA and insulin in these cells. In tRA‐treated cells, augmentation of the phosphorylation of protein tyrosine residue was accompanied by an increase in the activity of protein tyrosine kinase and was inhibited by actinomycin D, inhibiting cable growth. Activation of this enzyme in tRA‐treated cells probably depends on RNA synthesis. In insulin‐treated cells, augmentation of tyrosine residue phosphorylation occurred without any appreciable change in this enzyme’s activity and was hardly affected by actinomycin D. Phosphorylation of protein tyrosine residue seems to be activated by the binding of insulin to an insulin receptor. Pseudopodial cable growth in these cells treated with tRA or insulin was inhibited by wortmannin. Phosphatidylinositol 3 kinase probably participates in tRA and insulin signal transduction systems.
Endoderm differentiation and inductive effect of activin‐treated ectoderm in Xenopus
Authors: Ninomiya Hiromasa , Takahashi Shuji , Tanegashima Kousuke , Yokota Chika , Asashima Makoto
Published: March 2003

When presumptive ectoderm is treated with high concentrations of activin A, it mainly differentiates into axial mesoderm (notochord, muscle) in Xenopus and into yolk‐rich endodermal cells in newt (Cynops pyrrhogaster). Xenopus ectoderm consists of multiple layers, different from the single layer of Cynops ectoderm. This multilayer structure of Xenopus ectoderm may prevent complete treatment of activin A and subsequent whole differentiation into endoderm. In the present study, therefore, Xenopus ectoderm was separated into an outer layer and an inner layer, which were individually treated with a high concentration of activin A (100 ng/mL). Then the differentiation and inductive activity of these ectodermal cells were examined in explantation and transplantation experiments. In isolation culture, ectoderm treated with activin A formed endoderm. Ectodermal and mesodermal tissues were seldom found in these explants. The activin‐treated ectoderm induced axial mesoderm and neural tissues, and differentiated into endoderm when it was sandwiched between two sheets of ectoderm or was transplanted into the ventral marginal zone of other blastulae. These findings suggest that Xenopus ectoderm treated with a high concentration of activin A forms endoderm and mimics the properties of the organizer as in Cynops.
Sonic hedgehog signaling during digit pattern duplication after application of recombinant protein and expressing cells
Authors: Wada Naoyuki , Kawakami Yasuhiko , Nohno Tsutomu
Published: March 2003

HoxD expression and cartilage pattern formation were compared after application of a recombinant amino‐ terminal peptide of Sonic hedgehog protein (Shh‐N) and implantation of cells expressing the Sonic hedgehog (Shh) gene. During digit duplication after implantation of a Shh‐N‐soaked bead, BMP‐2 and Patched expression was transiently induced in the anterior limb mesenchyme 20 h after grafting, but was reduced to the basal level 48 h after grafting. On the contrary, when Shh‐expressing cells were grafted to the anterior limb bud, expression domains of the BMP‐2 and Patched genes were initially induced in the restricted region in close proximity to the grafted cells. Induced expression of BMP‐2 and Patched was maintained in the anterior‐peripheral region of the limb bud for 42 h after grafting. In either case, HoxD12 and HoxD13 were consistently induced in the anterior‐distal limb mesenchyme, accompanying mirror‐image duplication of the digit pattern. Induction and maintenance of HoxD expression were consistent with the resultant digit pattern. A steep gradient of Shh activity provided by Shh‐expressing cells is most adequate to induce complete digit pattern, as compared to the shallow gradient provided by Shh‐N protein released from a bead. These results suggest that positional identity is respecified by Shh‐N activity within the first 24 h during digit duplication, and that Shh‐N on its own is not acting as a long‐range signaling molecule to determine positional identity at a distance in the limb bud.
Spatial expression of Drosophila Glutathione S‐transferase‐D1 in the alimentary canal is regulated by the overlying visceral mesoderm
Authors: Nakamura Akira , Yoshizaki Izumi , Kobayashi Satoru
Published: March 2003

Regional gene expression within Drosophila gut epithelium is regulated by the homeotic genes expressed in the overlying visceral mesoderm. Here it is reported that Glutathione S‐transferase‐D1 (Gst‐D1) had three distinctive expression domains in the gut epithelia: the inner epithelium of the proventriculus, the anterior border of the hindgut epithelium, and the midgut epithelium. Gst‐D1 expression in the midgut epithelium became restricted to the region that later formed the third midgut constriction. This spatial restriction within the midgut epithelium required abdominal‐A activity in the overlying visceral mesoderm, suggesting that Gst‐D1 will be a useful marker to analyze the mechanism of gene regulation across the mesoderm and endoderm.
Changes in the adhesive properties of dissociated and reaggregated Xenopus laevis embryo cells
Authors: Kuroda Hiroki , Sakumoto Hirofumi , Kinoshita Kei , Asashima Makoto
Published: March 2003

Activin A is a member of the transforming growth factor β superfamily, and the strongest candidate mesoderm‐inducer. The initial adhesive property changes in amphibians are likely to be mediated by mesoderm‐inducers like activin A. The manner in which these changes actually occur, however, remains poorly understood. In the present study, the adhesive property changes mediated by activin A were directly demonstrated. Activin A functioned as a morphogen at low concentrations (less than 0.5 ng/mL), with no effect on the type A adhesive property. But at high concentrations (1 ng/mL), it induced another type of adhesive property, type N, and at very high concentrations (more than 10 ng/mL), it induced yet another type of adhesive property, type Y. Cells that have types A, N, and Y adhesive properties ultimately differentiated into atypical epidermis, notochord, and yolk‐rich cells, respectively. It was also shown that these changes occurred between 5 and 10 h after induction by activin A. The implications of these results for the relationship between the adhesive property acquired during early and later stages of differentiation are also discussed.
Role of type III iodothyronine 5‐deiodinase gene expression in temporal regulation of Xenopus metamorphosis
Authors: Kawahara Akira , Gohda Yoshitaka , Hikosaka Akira
Published: March 2003

To elucidate the role of type III iodothyronine 5‐deiodinase (5‐D) in the temporal regulation of amphibian metamorphosis, the regulation of gene expression of 5‐D and thyroid hormone receptor β (TRβ) in organs of Xenopus laevis was investigated. High levels of TRβ mRNA in the respective organs were observed at the times of their major morphological changes. Expression of the 5‐D gene was highly regulated among the organs during metamorphosis, including up‐regulation in the tail and down‐regulation in the liver. The tail and liver expressed 5‐D gene before their metamorphic changes. These precocious expressions correlated with the lower responsiveness to exogenously added triiodo‐L‐thyronine (T3) for inducing a high level of TRβ mRNA expression. However, the same organs responded to lower doses of T3 to regulate 5‐D gene expression as seen in spontaneous metamorphosis. The induction of 5‐D gene expression was considerably delayed in the intestine, even at an excess dose of T3. Thus, the two genes in a given organ appeared to respond to T3 either with different dose dependencies or with different timetables. The results obtained are also discussed in respect to recent findings in Rana catesbeiana.
Changes in the activities of protein phosphatase type 1 and type 2A in sea urchin embryos during early development
Authors: Kawamoto Manabu , Fujiwara Akiko , Yasumasu Ikuo
Published: March 2003

In the eggs and embryos of sea urchins, the activity of protein phosphatase type 2A (PP2A) increased during the developmental period between fertilization and the morula stage, decreased after the prehatching blastula stage and increased again after hatching. The PP2A activity changed keeping pace with alteration to the activities of cAMP‐dependent protein kinase (A kinase), Ca2+/calmodulin‐dependent protein kinase (CaM kinase) and casein kinase. Probably, PP2A contributes to the quick turning off of cellular signals because of protein phosphorylation. The activity of protein phosphatase type 1 (PP1) was not detectable up to the morula stage and appreciably increased thereafter. In the isolated nucleus fraction, specific activities of PP1 and PP2A were higher than in whole embryos at all stages in early development. Exponential increase in the number of nuclei because of egg cleavage probably makes PP1 activity detectable in whole embryos after the morula stage. In isolated nuclei, the activities of PP1 and PP2A appreciably decreased after hatching, whereas the activities of A kinase, Ca2+/phospholipid‐dependent protein kinase (C kinase) and CaM kinase, as well as casein kinase, became higher. In nuclei, cellular signals caused by protein phosphorylation after hatching do not seem to be turned off by these protein kinases so quickly as before hatching. The PP1 and PP2A in nuclei also seem to contribute to the elimination of signal noise.
Ectopic activation of the transcription promoter for the testis‐specific mouse Pgk‐2 gene on elimination of a cis‐acting upstream DNA region
Authors: Ando Hiroshi , Haruna Yoshiya , Suzuki Misao , Yamada Shuichi , Okabe Masaru , Nakanishi Yoshinobu
Published: March 2003

Transgenic mice carrying the coding sequence of β‐galactosidase, for which expression was driven by various upstream regions including the transcription promoter of the testis‐specific mouse Pgk‐2 gene, were generated. Expression of β‐galactosidase mRNA driven by the region between nucleotide positions – 1404 and + 61, with respect to the transcription initiation site numbered + 1, was examined by reverse transcription‐mediated polymerase chain reaction, blot hybridization and in situ hybridization, and compared with that of endogenous Pgk‐2 mRNA. The results revealed that the 1.4 kb DNA region is sufficient for determining the organ‐specific, developmental stage‐specific and spermatogenic stage‐specific transcription of the mouse Pgk‐2 gene. When the region between – 684 and + 61 was used to generate transgenic mice, β‐galactosidase mRNA was detectable not only in the testis, but also in other organs such as brain and lung. However, the timing and cell‐type specificity of testicular expression of β‐galactosidase mRNA were retained in these mice. Because the region between – 1404 and – 685 repressed the Pgk‐2 promoter in somatic cell‐derived cell lines, it is suggested that the organ specificity of Pgk‐2 transcription is achieved at least partly by negative regulation.
Disappearance of an epithelial cell surface‐specific glycoprotein (Epith‐1) associated with epithelial–mesenchymal conversion in sea urchin embryogenesis
Authors: Kanoh K. , Aizu G. , Katow H.
Published: March 2003

Cell surface modification during mesenchyme ingression was examined using a monoclonal antibody (mAb), anti‐Epith‐1 mAb, raised against a protein (Epith‐1) that was confined to the lateral surface of the epithelial cells in embryo of the sea urchin, Temnopleurus hardwicki. The mAb epitope was N‐glycosylated oligosaccharides of 160 kDa monomeric Epith‐1 protein. The glycoprotein was negatively charged, and its isoelectric point (IP) was 4.98. The mAb, however, is not immunologically cross‐reactive with other sea urchin embryos including Hemicentrotus pulcherrimus, Strongylocentrotus nudus, and Scaphechinus mirabilis. Epith‐1 is present initially in the cytoplasm of unfertilized eggs. Cytoplasmic Epith‐1 shifted to the cell surface to be integrated in plasma membrane during the first cleavage, and remained there during early embryogenesis by retaining the same relative molecular mass (Mr). During primary and secondary mesenchyme ingression periods, however, Epith‐1 disappears from the presumptive mesenchyme cell surface that was associated with internalization of the protein. In plutei, an additional anti‐Epith‐1 mAb‐positive protein appears at the 142 kDa region, which was not associated with any visible alteration of the histologic localization of the protein in larvae. Anti‐Epith‐1 mAb IgG did not inhibit the reaggregation of epithelial cells in vitro, which suggests that either the protein is not involved in cell–cell adhesion or that the mAb is not recognizing the active site of the protein.
Overexpression of Fyn tyrosine kinase causes abnormal development of primary sensory neurons in Xenopus laevis embryos
Authors: Saito Rika , Fujita Naoko , Nagata Saburo
Published: March 2003

The expression and function of the Src family protein tyrosine kinase Fyn in Xenopus laevis embryos have been examined. In situ hybridization analysis demonstrated nervous system‐specific expression of Fyn mRNA in tail‐bud embryos. However, a class of primary sensory neurons; that is, Rohon–Beard (RB) neurons, which is positive for immunoglobulin superfamily cell adhesion molecules (CAM), neural cell adhesion molecule (N‐CAM) and contactin, is devoid of Fyn expression. Injection of Fyn mRNA into one of the blastomeres at the 2‐cell stage led to overexpression of Fyn in the injected half of the tail‐bud embryos. Immunolabeling of the embryos with anti‐HNK‐1 antibody revealed that the peripheral axons of RB neurons were partially misguided and bound to each other to form abnormal subcutaneous fascicles. Similar abnormality was induced by injection of the Fyn overexpression vector. The incidence of abnormality appeared dose‐dependent, being 68–92% of the injected embryos at 50–400 pg of mRNA. Co‐injection of the contactin antisense vector depleted contactin mRNA accumulation without affecting Fyn overexpression and reduced the incidence of the abnormal RB‐cell phenotype. However, the N‐CAM antisense was ineffective in reducing this abnormality. These results suggest that Fyn can modify signals regulating axonal guidance or fasciculation in the developing X. laevis nervous system and that contactin may affect this action of Fyn.
Spatial expression patterns of genes involved in cyclic AMP responses in Dictyostelium discoideum development
Authors: Tsujioka Masatsune , Yokoyama Masako , Nishio Keiko , Kuwayama Hidekazu , Morio Takahiro , Katoh Mariko , Urushihara Hideko , Saito Tamao , Ochiai Hiroshi , Tanaka Yoshimasa , Takeuchi Ikuo , Maeda Mineko
Published: March 2003

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G‐protein α2 and β subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA‐C) and cAMP phosphodiesterases (PDE and REG‐A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal‐regulated kinase‐2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA‐C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis.
Effects of microgravity on the larval development, metamorphosis and reproduction of the urodele amphibian Pleurodeles waltl
Authors: Dournon Christian , Durand Danielle , Tankosic Christiane , Membre Hervè , Gualandris‐Parisot Lydie , Bautz Alain
Published: March 2003

The FERTILE experiment was twice performed onboard the Mir space station during the Cassiopée and Pégase French space missions. The goal was to analyze the effects of microgravity on fertilization and embryonic development, and then on further development on the ground in the amphibian Pleurodeles waltl. The present paper reports development that occurred in the laboratory after landing. Recovered on the ground at the hatching stage, young larvae reared at room temperature underwent metamorphosis and became adults without obvious abnormalities. Of particular interest was the rearing temperature that induced a delayed metamorphosis for animals from the Cassiopée space mission, but not for animals from the Pégase mission. The rate of development and the morphology were analogous in these animals and in ground controls reared in a similar annual period. Analysis of offspring was performed using these animals. Males born in space were first mated with control ground‐born females and then with females born in space. The mating gave progeny that developed normally. Depending on the methods used and on the limits of the analyses, the results clearly demonstrated that animals born in space were able to live and reproduce after return to the ground.
Activation of the maternally preset program of apoptosis by microinjection of 5‐aza‐2′‐deoxycytidine and 5‐methyl‐2′‐deoxycytidine‐5′‐triphosphate in Xenopus laevis embryos
Authors: Kaito Chikara , Kai Masatake , Higo Takayasu , Takayama Eiji , Fukamachi Hiroshi , Sekimizu Kazuhisa , Shiokawa Koichiro
Published: March 2003

The present study examines the effects on embryogenesis of microinjecting Xenopus laevis fertilized eggs with 5‐aza‐2′‐deoxycytidine (5‐Aza‐CdR), which induces hypomethylation of DNA, and 5‐methyl‐2′‐ deoxycytidine‐5′‐triphosphate (5‐methyl‐dCTP), which induces hypermethylation of DNA. Embryos injected with either one of these analogs cleaved normally until the mid‐blastula stage, but underwent massive cell dissociation and stopped development at the early gastrula stage. Dissociated cells that appeared here were positive by terminal deoxyribonucleotidyl transferase‐mediated deoxyuridine triphosphate–digoxigenin nick end‐labeling and contained fragmented nuclei with condensed chromatin. The DNA from these cells formed a ‘ladder’ on electrophoresis. Furthermore, the induction of cell dissociation by 5‐Aza‐CdR and 5‐methyl‐dCTP was postponed by 2–3 h by co‐injection of Bcl‐2 mRNA and the normal metabolite (CdR and dCTP, respectively). Using a specific antibody against 5‐methyl‐cytosine, we confirmed that 5‐Aza‐CdR induces hypomethylation, whereas 5‐methyl‐dCTP induces hypermethylation in X. laevis embryos before the onset of cell dissociation. Incorporation of radioactive precursors revealed that synthesis of DNA, and also RNA, is inhibited significantly in both 5‐Aza‐CdR‐injected and 5‐methyl‐dCTP‐injected embryos. These results show that 5‐Aza‐CdR and 5‐methyl‐dCTP are incorporated into DNA and induce apoptosis, probably through alteration of DNA methylation coupled with inhibition of DNA replication and/or transcription.
Involvement of a urethane‐sensitive system in timing the onset of gastrulation in Xenopus laevis embryos
Authors: Kuroda Satoshi , Satoh Takashi , Shinagawa Atsunori
Published: March 2003

This paper describes success in delaying the onset of gastrulation in Xenopus laevis embryos without damage to their subsequent development by temporarily arresting cleavage with urethane. Exposure of X. laevis embryos to 150 mM urethane before gastrulation resulted in cleavage arrest and its removal led to cleavage resumption. During cleavage arrest, cyclic activities including nuclear replication and the M‐phase‐promoting factor cycle continued, although their duration was lengthened to nearly 1.8‐fold that of the controls. Because of a 30‐min time lag from removal of urethane to resumption of cleavage, as well as the retardation of cyclic activities during cleavage arrest, the development of embryos after a 60‐min exposure to urethane lagged two cell cycles behind that of control embryos. Here, the two cell cycle delay is equivalent to 50 min at 22–23°C. The start of gastrulation in exposed embryos was accordingly delayed about 50 min, although the delay in mid‐blastula transition was as little as 20–25 min. Consistent results were obtained in embryos exposed to urethane for 90 or 120 min and those exposed to procaine or NH4Cl for 60 min. Although these results imply that delay in the start of gastrulation in exposed embryos is ascribed simply to delay in their development raised by cleavage arrest, at the same time they suggest that the onset of gastrulation is timed by systems sensitive to urethane, procaine and NH4Cl in X. laevis embryos.
Inhibitors of procollagen C‐terminal proteinase block gastrulation and spicule elongation in the sea urchin embryo
Authors: Huggins Luke G. , Lennarz William J.
Published: March 2003

In the sea urchin embryo, inhibition of collagen processing and deposition affects both gastrulation and embryonic skeleton (spicule) formation. It has been found that cell‐free extracts of gastrula‐stage embryos of Strongylocentrotus purpuratus contain a procollagen C‐terminal proteinase (PCP) activity. A rationally designed non‐peptidic organic hydroxamate, which is a potent and specific inhibitor of human recombinant PCP (FG‐HL1), inhibited both the sea urchin PCP as well as purified chick embryo tendon PCP. In the sea urchin embryo, FG‐HL1 inhibited gastrulation and blocked spicule elongation, but not spicule nucleation. A related compound with a terminal carboxylate rather than a hydroxamate (FG‐HL2) did not inhibit either chick PCP or sea urchin PCP activity in a procollagen‐cleavage assay. However, FG‐HL2 did block spicule elongation without affecting spicule nucleation or gastrulation. Neither compound was toxic, because their effects were reversible on removal. It was shown that the inhibition of gastrulation and spicule elongation were independent of tissue specification events, because both the endoderm specific marker Endo1 and the primary mesenchyme cell specific marker SM50 were expressed in embryos treated with FG‐HL1 and FG‐HL2. These results suggest that disruption of the fibrillar collagen deposition in the blastocoele blocks the cell movements of gastrulation and may disrupt the positional information contained within the extracellular matrix, which is necessary for spicule formation.
Morphologic and functional development of whole human fetal stomachs grafted into nude mice
Authors: Angioi Karine , Hatier Renée , Muhale Filipe , Plénat François , Duprez Adrien
Published: March 2003

To study in vivo the cellular differentiation and secretion of human developing fetal stomach, ethically and technically impossible to perform in utero, 256 fetal stomachs were xenografted. Human stomachs from 6‐ to 10‐week‐old fetuses were grafted for 1–273 days into nude mice. Biopsies for immunohistochemistry, hybridization and electron microscopy were taken and a catheter introduced into the human stomach. Macroscopic growth was fast and cells in S phase were numerous during the first 9 weeks, then the stomach size was stable and the gastric mucosa, of adult type, remained normal. In situ hybridization detected only a minute mouse mesenchymal chimerism in the graft. Chromogranin A, intrinsic factor and H+/K+ adenosine triphosphatase were immunohistolocally detected in epithelial cells 20 days after grafting, gastrin was detected after 30 days and pepsinogen after 60 days. The pH in gastric juice, which was at 8.0 ± 0.1 from days 10–25, dropped from 4.39 ± 1.80 at 30 days to 1.58 ± 0.29 at 90 days. Intrinsic factor was stable and pepsin ranged from 6.8 ± 7.8 to 134 ± 51 units at 90 days. The differentiation of the epithelial cells in xenografts was very accelerated in comparison to that in utero.
Calmodulin binds to inv protein: Implication for the regulation of inv function
Authors: Yasuhiko Yukuto , Imai Fumiyasu , Ookubo Kenshi , Takakuwa Yuichi , Shiokawa Koichiro , Yokoyama Takahiko
Published: March 2003

Establishment of the left–right asymmetry of internal organs is essential for the normal development of vertebrates. The inv mutant in mice shows a constant reversal of left–right asymmetry and although the inv gene has been cloned, its biochemical and cell biological functions have not been defined. Here, we show that calmodulin binds to mouse inv protein at two sites (IQ1 and IQ2). The binding of calmodulin to the IQ2 site occurs in the absence of Ca2+ and is not observed in the presence of Ca2+. Injection of mouse inv mRNA into the right blastomere of Xenopus embryos at the two‐cell stage randomized the left–right asymmetry of the embryo and altered the patterns of Xnr‐1 and Pitx2 expression. Importantly, inv mRNA that lacked the region encoding the IQ2 site was unable to randomize left–right asymmetry in Xenopus embryos, implying that the IQ2 site is essential for inv to randomize left–right asymmetry in Xenopus. These results suggest that calmodulin binding may regulate inv function. Based on our findings, we propose a model for the regulation of inv function by calcium–calmodulin and discuss its implications.
Role of the thrombopoietin (TPO)/Mpl system: c‐Mpl‐like molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis
Authors: Kakeda Minoru , Kyuno Jun‐ichi , Kato Takashi , Nishikawa Mitsuo , Asashima Makoto
Published: March 2003

Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c‐Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)‐4 and increased the number of both erythrocytes and leukocytes in a dose‐dependent manner. Addition of anti‐c‐Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood‐like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c‐Mpl‐like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal– lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c‐Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c‐Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.
A novel Dictyostelium Cdk8 is required for aggregation, but is dispensable for growth
Authors: Takeda Kosuke , Saito Tamao , Ochiai Hiroshi
Published: March 2003

When Dictyostelium cells starve, they express genes necessary for aggregation. Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation and that forms small plaques on bacterial lawns, thus indicating slow growth. Sequencing of the mutated locus showed a strong similarity to the catalytic domain of cdc2‐related kinase genes. Phylogenetic analysis further indicated that the amino acid sequence was more close to cyclin‐dependent kinase 8 than to the sequence of other cyclin‐dependent kinases. Thus, we designated this gene as Ddcdk8. The Ddcdk8‐null cells do not aggregate and grow somewhat more slowly than parental cells when being shaken in axenic medium or laid on bacterial plates. To confirm whether these defective phenotypes were caused by disruption of this gene, the Ddcdk8‐null cells were complemented with DdCdk8 protein expressed from an endogenous promoter, but not an actin promoter, and when the complemented cells were then allowed to grow on a bacterial lawn, they began to aggregate as the food supply was depleted and finally became fruiting bodies. The results suggest that properly regulated DdCdk8 activity is essential for aggregation. Because, when starved, Ddcdk8‐null cells do not express the acaA transcripts required for aggregation, we deduce that Ddcdk8 is epistatic for acaA expression, indicating that the DdCdk8 products may regulate expression of acaA and/or other genes.
Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos
Authors: Iraha Fumie , Saito Yoshinari , Yoshida Keiko , Kawakami Masatoki , Izutsu Yumi , Daar Ira Owen , Maéno Mitsugu
Published: March 2003

In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie‐2, a Xenopus homolog of the tie‐2 receptor tyrosine kinase, was examined. Whole‐mount in situ hybridization analysis revealed that Xtie‐2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie‐2‐positive cells are predominantly present just outside the boundary of α‐globin‐positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie‐2 and α‐globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a ‘dorsal determinant’ from the vegetal cytoplasm at the 1‐cell stage. Second, expression of both Xtie‐2 and α‐globin were markedly induced in ectodermal explants (animal caps) from embryos co‐injected with activin and bone morphogenetic protein (BMP)‐4 RNA. Furthermore, both Xtie‐2 and α‐globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP‐4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP‐4‐dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.
Variations in abiotic defense within myrmecophytic and non‐myrmecophytic species of Macaranga in a Bornean dipterocarp forest
Authors: Nomura Masahiro , Itioka Takao , Itino Takao
Published: March 2003

We examined the interspecific variations in intensity of total abiotic (chemical and physical) defenses in five sympatric Macaranga (Euphorbiaceae) species, including three myrmecophytic species. The intensity of the total abiotic defense for each Macaranga species was estimated by measuring inhibiting effects on the growth performance of the common cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) when the cutworm larvae were fed fresh leaves of each Macaranga species. Indices of the growth performance, number of dead larvae, pupal weight and length of larval period were obtained. We found that the intensities of total abiotic defense of the two non‐myrmecophytic species were significantly stronger than those of the three myrmecophytic species, and that there was a significant difference in intensity even within the three myrmecophytic species. The former result supports the hypothesis that, unlike non‐myrmecophytic species, myrmecophytic species cannot invest so many metabolic resources in abiotic defense, because they have to allocate nutrients to biotic defense (toward biotic defense agents). Moreover, the latter result suggests the possibility that the three sympatric myrmecophytes have different defense strategies, with a trade‐off between abiotic and biotic defense, and/or with a trade‐off between defense and other life‐history traits such as growth and reproduction. Abiotic defense can be roughly separated into physical and chemical mechanisms. To assess the intensity of the physical defense of Macaranga leaves, we measured the leaf toughness of each species. In addition, to assess the intensity of the plant’s general chemical defense, cutworm larvae were reared on an artificial diet containing dry leaf powder of each Macaranga species, and their growth performances were compared. The estimated orders of intensity of both leaf toughness and general chemical defense coincided with that of the total abiotic factors measured by the growth performance of cutworm on fresh leaves. This suggests the presence of both physical defenses, represented by leaf toughness, and a general chemical defense affecting the intensity of the total abiotic defense in similar ways.
Seed dormancy/germination traits of seven Persicaria species and their implication in soil seed‐bank strategy
Authors: Araki Sachiko , Washitani Izumi
Published: March 2003

In order to predict species‐specific potential to form persistent soil seed‐banks and to characterize the dynamics of their seed‐banks, the seed dormancy/germination traits of seven Persicaria (Polygonum s.lat.) species sharing lakeshore habitats in central Japan were examined. Strict light requirements for seed germination were not observed in any of the species examined. Although all species required moist chilling (0–6 weeks) to break seed dormancy and were sensitive to temperature fluctuation, the degree of both responses varied between species. Seed germination of Persicaria hydropiper (L.) Spach, Persicaria lapathifolia (L.) S.F. Gray, and Persicaria longiseta (De Bruyn) Kitag. was more accelerated by temperature fluctuation and required shorter chilling periods compared with Persicaria japonica (Meisn.) H. Gross, Persicaria maackiana (Regel) Nakai, Persicaria thunbergii (Sieb. et Zucc.) H. Gross, and Persicaria sieboldi (Maisn.) Onki. Secondary dormancy was induced in all species at higher temperatures (24 and 30°C). A persistent seed‐bank strategy suggested by the dormancy/germination traits of the studied species was also demonstrated by seedling emergence from surface soils collected from the natural habitat immediately before seed dispersal, as well as by viable seed persistence for 13 months in the field in a seed burial experiment. In the natural habitat, the species with longer chilling requirements occurred in various microhabitats, including the interior of moist tall grasslands, whereas the species having higher sensitivity to temperature fluctuation were most frequently found in sparsely vegetated microhabitats.
Photosynthesis and water‐relation traits of the summer annual C4 grasses, Eleusine indica and Digitaria adscendens, with contrasting trampling tolerance
Authors: Kobayashi Tsuyoshi , Hori Yoshimichi
Published: March 2003

Two summer annual C4 grasses with different trampling susceptibilities were grown as potted plants, and diurnal leaf gas exchange and leaf water potential in each grass were compared. The maximum net photosynthetic rate, leaf conductance and transpiration rate were higher in the trampling‐tolerant Eleusine indica (L.) Gaertn. than in trampling sensitive Digitaria adscendens (H. B. K.) Henr. Leaf water potential was much lower in E. indica than in D. adscendens. There were no differences in soil‐to‐leaf hydraulic conductance and leaf osmotic potential at full turgor as obtained by pressure–volume analysis. However, the bulk modulus of elasticity in cell walls was higher in E. indica leaves than in D. adscendens leaves. This shows that the leaves of E. indica are less elastic. Therefore, the rigid cell walls of E. indica leaves reduced leaf water potential rapidly by decreasing the leaf water content, supporting a high transpiration rate with high leaf conductance. In trampled habitats, such lowering of leaf water potential in E. indica might play a role in water absorption from the compacted soil. In contrast, the ability of D. adscendens to colonize dry habitats such as coastal sand dunes appears to be due to its lower transpiration rate and its higher leaf water potential which is not strongly affected by decreasing leaf water content.
Variation of sterility and fertility in Utricularia australis f. australis in Hokkaido, northern Japan
Authors: Araki Satoru
Published: March 2003

Seed‐production ability was studied in Utricularia australis R. Br. f. australis Komiya and Shibata through field observation, pollination experiment, pollen culture, and isozyme analysis for populations in various regions of Hokkaido, northern Japan. Utricularia australis f. australis has previously been postulated to be sterile in Japan, however, in the present study, fertile populations were found in eastern Hokkaido. Bisexual sterility and male sterility were suspected to be present in populations in the western part of Hokkaido. There was also a strain that seemed to produce no seed because of intra‐clonal inbreeding depression or self‐incompatibility. Isozyme analysis of three enzyme systems indicated a uniformity of multienzymatic phenotype (MEP) in each population or region. The MEP grouping of populations corresponded to the type of sterility and fertility revealed by pollination and pollen culture experiments. These experiments suggested that each regional strain had its own sterility or fertility type.
Winter CO2 flux from soil and snow surfaces in a cool‐temperate deciduous forest, Japan
Authors: Mariko Shigeru , Nishimura Noboru , Mo Wenhong , Matsui Yoshisuke , Kibe Takeshi , Koizumi Hiroshi
Published: March 2003

We measured diurnal and wintertime changes in CO2 fluxes from soil and snow surfaces in a Japanese cool‐temperate Quercus/Betula forest between December 1994 and May 1995. To evaluate the relationship between these winter fluxes and temperature, flux measurements were made with the open‐flow infrared gas analyzer (IRGA) method rather than with the more commonly used closed chamber method or the snow CO2 profile method. The open‐flow IRGA method proved to be more successful in measurements of winter CO2 fluxes than the two standard methods. Despite colder air temperatures, soil temperature profiles were greater than 0°C because of the thermal insulation effect of deep snowpack. This reveals that soil temperature is satisfactory for microbial respiration throughout the winter. Unfrozen soils under the snowpack showed neither diurnal nor wintertime trends in CO2 fluxes or in soil surface temperature, although there was a daily snow surface CO2 flux of 0.18–0.32 g m−2. By combining this with other reference data, Japanese cool‐temperate forest soils in snowy regions can be estimated to emit < 100 g m−2 carbon over an entire winter, and this value accounts for < 15% of the annual emission. In the present study, when data for all winter fluxes were taken together, fluxes were most highly correlated with deep soil temperatures rather than the soil surface temperature. Such a high correlation can be attributed to the relatively increased respiration of the deep soil where the temperature was higher than the soil surface temperature. Thus, deeper soil temperature is a better predictor of winter CO2 fluxes in cold and snowy ecosystems.