bluuebunny/crossref_metadata_2025 huggingface dataset:
The guinea‐pig oesophagus is a versatile in vitro preparation for pharmacological studies
Authors: Kerr Karen P
Published: March 2003

1. The isolated oesophagus of the guinea‐pig is a useful preparation that can be used as an alternative to the phrenic nerve–diaphragm preparations that have been used traditionally in the determination of the actions of drugs and toxins at the neuromuscular junction. The guinea‐pig isolated oesophagus can also provide information about effects at ganglionic nicotinic receptors, which are not present in the diaphragm preparations.

2. The muscularis externa of the body of the oesophagus consists exclusively of striated muscle fibres. The myenteric plexus is found between the outer longitudinally arranged layer and the inner circular layer. The muscularis mucosae contains smooth muscle fibres arranged longitudinally.

3. The vagal nerves are comprised of special vagal efferents that innervate the striated muscle fibres directly and ‘parasympathetic’ vagal fibres that synapse in the myenteric ganglia and, subsequently, affect the smooth muscle of the muscularis mucosae. Thus, both striated and smooth muscle responses to vagal nerve stimulation can be studied. In addition, afferent neurons run in the vagus.

4. Studies using various isolated oesophageal preparations have been reviewed. These consist of the whole oesophagus (including striated muscle, myenteric plexus and smooth muscle), the isolated mucosal layer (striated muscle and the myenteric plexus both absent) and the whole oesophagus with the vagus nerve attached. Comparative studies of the effects of drugs acting directly on the muscularis mucosae and/or indirectly via the intramural plexuses can be performed using the whole oesophagus and the isolated mucosal layer.

5. The use of the guinea‐pig isolated vagus nerve–oesophagus preparation allows potency determinations for both neuromuscular and ganglion blockade of various non‐depolarizing muscle relaxants to be performed simultaneously under identical conditions. Furthermore, the phenomenon of fade, a waning of tetanic tension during the stimulation period, can be studied.

6. A potential application of this preparation is the simultaneous screening of the constituents of snake venoms for activity at the neuromuscular junction and/or the ganglion. It is also suggested that new calcium channel blockers could be screened in this preparation because different voltage‐sensitive calcium channels are involved in neurotransmitter release from nerve terminals at the neuromuscular junction and autonomic cholinergic neuroeffector sites.
Changes in mRNA expression induced by sustained noradrenaline stimulation are different for α1‐adrenoceptor subtypes in HEK293 cells
Authors: Lei Beilei , Zhang Youyi , Han Chide
Published: March 2003

1. The aim of the present study was to investigate noradrenaline (NA)‐induced regulation of α1‐adrenoceptor (AR) mRNA expression in human embryonic kidney (HEK) 293 cells stably expressing cloned α1‐AR subtypes with similar receptor densities. Stable transfection was performed by calcium phosphate precipitation. Receptor expression was detected by radioligand binding assay. The mRNA expression was measured by RNase protection assay.

2. α1‐Adrenoceptor subtype mRNA respond in distinct ways following prolonged exposure to NA. The mRNA level of the α1A‐AR subtype was unchanged, the mRNA level of the α1B‐AR subtype was increased and the mRNA level of the α1D‐AR subtype declined time dependently. The protein kinase C (PKC) inhibitor calphostin C or RO 31‐8220 abolished the NA‐induced downregulation of α1D‐AR mRNA. Phorbol myristate acetate (PMA), a PKC activator, similarly repressed the effects of NA on α1D‐AR. However, calphostin C, RO 31‐8220 or PMA had no effect on the induction of α1B‐AR mRNA by NA. The Ca2+‐ATPase inhibitor thapsigargin or the calcium chelator 1,2‐bis‐(o‐aminophenoxy) ethane‐N,N,N′,N′‐tetraacetic acid tetra (acetoxymethyl) ester (BAPTA/AM) had no effect on the repression of α1D‐AR mRNA, but did inhibit the induction of α1B‐AR mRNA by NA. Noradrenaline significantly decelerated the degradation of α1B‐AR mRNA, but had no effect on the degradation of α1D‐AR mRNA.

3. Thus, the mRNA expression of three α1‐AR subtypes in HEK293 cells is differentially regulated through distinct signal transduction pathways under sustained NA stimulation. The upregulation of α1B‐AR mRNA is via the Ca2+ pathway, whereas the downregulation of α1D‐AR mRNA is via the PKC pathway.
Effect of Undaria pinnatifida (Wakame) on the development of cerebrovascular diseases in stroke‐prone spontaneously hypertensive rats
Authors: Ikeda Katsumi , Kitamura Akiko , Machida Hiroko , Watanabe Miyuki , Negishi Hiroko , Hiraoka Junko , Nakano Takahisa
Published: March 2003

1. We showed that a nutritional factor was able to attenuate the development of hypertension and its related diseases in stroke‐prone spontaneously hypertensive rats (SHRSP). In the present study, the effect of Wakame, an edible brown seaweed, on the development of stroke was examined in SHRSP.

2. We studied the treatment with 5% (w/w in a diet) Wakame powder in salt‐loaded (0.5% NaCl in drinking water) SHRSP. Salt‐loaded animals treated with 5% cellulose or kaolin were used as controls. Wakame significantly delayed the development of stroke signs (P < 0.05) and significantly improved the survival rate of salt‐loaded SHRSP (P < 0.05). There was no significant difference in the elevation of blood pressure among the three groups during the observation period.

3. We isolated fucoxanthin, a carotinoid, from Wakame powder and studied its preventive effect on ischaemic cultured neuronal cell death. Fucoxanthin significantly attenuated neuronal cell injury in hypoxia and re‐oxygenation (P < 0.05).

4. Based on these results, we conclude that Wakame has a beneficial effect on cerebrovascular diseases in SHRSP, independent of hypertension. It is possible that fucoxanthin in Wakame may have a preventive effect against ischaemic neuronal cell death seen in SHRSP with stroke.
Effect of 6‐hydroxydopamine on gluconeogenesis in the rat renal cortex
Authors: Cojocel C , Thomson MS
Published: March 2003

1. In the present study, the effects of 6‐hydroxydopamine (6‐OHDA) on renal gluconeogenesis were investigated in vitro using rat renal cortical slices. Cisplatin, a known nephrotoxin, was used as a positive control. The working hypothesis for the present study was that 6‐OHDA, as a reactive oxygen species‐producing agent, could inhibit renal gluconeogenesis.

2. 6‐Hydroxydopamine is used for chemical sympathectomy because it selectively destroys adrenergic nerve endings. Long‐term use of levodopa causes a variety of side‐effects in parkinsonian patients. 6‐Hydroxydopamine has been reported to be present in the urine of parkinsonian patients on levodopa medication. The renal toxicity of endogenously formed 6‐OHDA is a matter of concern in these patients.

3. In one series of experiments, renal cortical slices were incubated for 60 min in medium containing 0.5, 1.0, 2.08, 5.15, 10.30 or 20.60 mg/mL 6‐OHDA at 37°C under a 100% O2 atmosphere. In another series of experiments, renal cortical slices were incubated in medium containing 10.30 mg/mL 6‐OHDA for 15, 30, 45, 60, 90 or 120 min or in 6‐OHDA‐free medium.

4. In a second series of experiments, renal cortical slices were incubated for 60 min in medium containing 0.25, 0.50, 0.75, 1.0, 1.25 or 1.50 mg/mL cisplatin at 37°C under a 100% O2 atmosphere. In another set of experiments, renal cortical slices were incubated in medium containing 1 mg/mL cisplatin for 15, 30, 45, 60, 90 or 120 min or in a cisplatin‐free medium.

5. The results of the studies in which slices were incubated in 6‐OHDA‐containing media indicate that 6‐OHDA induced a time‐ and concentration‐dependent decrease in renal gluconeogenesis. Therefore, 6‐OHDA causes functional injury of renal proximal tubule cells responsible for renal gluconeogenesis, thus leading to nephrotoxicity.
Upregulation of muscarinic receptors by long‐term nitric oxide inhibition in the rat ileum
Authors: Bricola Alice AO , Teixeira Simone A , De Luca Iara MS , Muscará Marcelo N , Abdala Fernando MF , Porto Catarina S , Zanesco Angelina , Antunes Edson , De Nucci Gilberto
Published: March 2003

1. The aim of the present study was to examine the effects of long‐term nitric oxide (NO) blockade on contractions of the rat ileum induced by muscarinic agonists.

2. Male Wistar rats received the NO synthesis inhibitor NG‐nitro‐l‐arginine methyl ester (l‐NAME; 20 mg/rat per day) in drinking water for 7, 15, 30 and 60 days. Concentration–responses curves to methacholine and carbachol were obtained and pEC50 values were calculated. Saturation binding assays were performed in membranes prepared from rat ileum after 60 days of l‐NAME treatment and the dissociation constant (KD) and maximal number of binding sites (Bmax) were determined by Scatchard analysis.

3. The NO synthase activity of the ileum was markedly reduced in all l‐NAME‐treated groups. At 60 days after l‐NAME treatment, a significant increase in the potency of methacholine (fourfold) and carbachol (threefold) was observed. In binding studies, we found a significant increase in Bmax for [3H]‐quinuclidinyl benzilate of approximately 57% in the l‐NAME treated group without any significant change in KD values. The contractile response to methacholine was not modified by the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo‐[4,3‐a]quinoxalin‐1‐one (3 µmol/L). No morphological alterations in the rat ileum were observed in l‐NAME‐treated rats.

4. Our findings suggest that treatment with l‐NAME for 60 days induces a marked increase in the potency of methacholine and carbachol, as well as an increase in receptor number in the rat ileum.
A calcium‐binding motif in SPARC/osteonectin inhibits chordomesoderm cell migration during Xenopus laevis gastrulation: Evidence of counter‐adhesive activity in vivo
Authors: Huynh My‐Hang , Helene Sage E. , Ringuette Maurice
Published: March 2003

Secreted protein, acidic, rich in cysteine (SPARC) is a Ca2+‐binding, counter‐adhesive, extracellular glycoprotein associated with major morphogenic events and tissue remodeling in vertebrates. In Xenopus laevis embryos, SPARC is expressed first by dorsal mesoderm cells at the end of gastrulation and undergoes complex, rapid changes in its pattern of expression during early organogenesis. Another study has reported that precocious expression of SPARC by injection of native protein into the blastocoele cavity of pregastrula embryos leads to a concentration‐dependent reduction in anterior development. Thus, normal development requires that the timing, spatial distribution, and/or levels of SPARC be regulated precisely. In a previous study, we demonstrated that injection of a synthetic peptide corresponding to the C‐terminal, Ca2+‐binding, EF‐hand domain of SPARC (peptide 4.2) mimicked the effects of native SPARC. In the present investigation, peptide 4.2 was used to examine the cellular and molecular bases of the phenotypes generated by the aberrant presence of SPARC. Exposure of late blastula embryos to LiCl also generated a concentration‐dependent reduction in anterior development; therefore, injections of LiCl were carried out in parallel to highlight the unique effects of peptide 4.2 on early development. At concentrations that caused a similar loss in anterior development (60–100 ng peptide 4.2 or 0.25–0.4 μg LiCl), LiCl had a greater inhibitory effect on the initial rate of chordomesoderm cell involution, in comparison with peptide 4.2. However, as gastrulation progressed, peptide 4.2 had a greater inhibitory effect on prospective head mesoderm migration than that seen in the presence of LiCl. Moreover, peptide 4.2 and LiCl had distinct influences on the expression pattern of dorso‐anterior markers at the neural and tail‐bud stages of development. Scanning electron microscopy showed that peptide 4.2 inhibited spreading of migrating cells at the leading edge of the involuting chordomesoderm. While still in close proximity to the blastocoele roof, many of the cells appeared rounded and lacked lamellipodia and filopodia extended in the direction of migration. In contrast, LiCl had no effect on the spreading or shape of involuting cells. These data are the first evidence of a counter‐adhesive activity for peptide 4.2 in vivo, an activity demonstrated for both native SPARC and peptide 4.2 in vitro.
Expression pattern, subcellular localization and structure–function relationship of rat Tpx‐1, a spermatogenic cell adhesion molecule responsible for association with Sertoli cells
Authors: Maeda Tomoji , Nishida Jun , Nakanishi Yoshinobu
Published: March 2003

The gene for a testicular cell adhesion protein called Tpx‐1, which mediates the binding of spermatogenic cells to Sertoli cells of the rat in primary culture, was previously cloned. Here the characterization of Tpx‐1 is reported. Tpx‐1 messenger ribonucleic acid (mRNA) became detectable in pachytene spermatocytes and continued to be present throughout development into elongated spermatids, while the amount of Tpx‐1 protein seemed to increase some time after the increment of mRNA. Tpx‐1 protein was also present, although less abundantly, in spermatozoa prepared from the epididymis. Tpx‐1 contains a cluster of hydrophobic amino acid residues near the amino terminus and a cysteine‐rich region in the carboxyl‐terminal half. Tpx‐1 fused with green fluorescence protein was secreted into the medium when expressed in a cultured cell line, depending on the presence of the amino‐terminal hydrophobic region. Moreover, Tpx‐1 was present in the medium of testicular cell primary culture. Structure–function analysis revealed that the amino‐terminal 101 amino acid residues were sufficient for cell adhesion activity, whereas the carboxyl‐terminal cysteine‐rich region was dispensable. In conclusion, Tpx‐1 is produced and secreted from spermatogenic cells at various differentiation stages, and mediates the interaction of those cells with Sertoli cells.
Application of efficient and specific gene transfer systems and organ culture techniques for the elucidation of mechanisms of epithelial– mesenchymal interaction in the developing gut
Authors: Fukuda Kimiko , Sakamoto Nobuyuki , Narita Tomohiro , Saitoh Kanako , Kameda Takashi , Iba Hideo , Yasugi Sadao
Published: March 2003

Epithelial–mesenchymal interactions are very important in the development of the vertebrate gut. In the avian embryonic stomach (proventriculus), expression of embryonic chick pepsinogen (ECPg) gene, which is specific to developing glandular cells in stomach epithelium, is regulated by mesenchymal influence. Molecular mechanisms of tissue‐specific transcriptional regulation of the ECPg gene and the molecular nature of the mesenchymal signals were analyzed using a combination of the classic organ culture system and gene transfer strategies. In the present review, three methods for the introduction of DNA into tissues are described: lipofection, electroporation and retroviral infection, and characteristics of each system are discussed.
Non‐dependence of cyclin E/Cdk2 kinase activity on the initiation of oocyte maturation in goldfish
Authors: Yoshida Noriyuki , Yamashita Masakane
Published: March 2003

Cdk2 kinase activity increases during oocyte maturation but neither cyclin A nor B is associated with Cdk2 in mature oocytes in goldfish. As a potential Cdk2 partner in meiosis, a cyclin E homolog was isolated from a goldfish oocyte cDNA library. A monoclonal antibody was raised against bacterially produced full‐length goldfish cyclin E. Both cyclin E and Cdk2 were already present in immature oocytes and their protein levels did not change remarkably during oocyte maturation. Cyclin E formed a complex mainly with Cdk2 just at the time of germinal vesicle breakdown (GVBD) in association with the increase in Cdk2 kinase activity, although a fraction of cyclin E bound to Cdk(s) other than Cdk2 and Cdc2. Ectopic activation of cyclin E/Cdk2 by the injection of cyclin E messenger RNA (mRNA) into immature oocytes did not induce maturation‐promoting factor (MPF) activation and GVBD. Furthermore, inhibition of cyclin E/Cdk2 kinase activity by the injection of p21SDI1 into the oocytes treated with 17α,20β‐dihydroxy‐4‐pregnen‐3‐one had no effect on MPF activation and GVBD. These results indicate that cyclin E/Cdk2 kinase activity is insufficient and unnecessary for initiating goldfish oocyte maturation.
Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin
Authors: Utoh Rie , Asahina Kinji , Suzuki Kenichi , Kotani Kaori , Obara Masanobu , Yoshizato Katsutoshi
Published: March 2003

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of α1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the α1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.
Modeling the model organism Dictyostelium discoideum
Authors: Nagano Seido
Published: March 2003

The cellular slime mold Dictyostelium discoideum is a fascinating organism, not only for biologists, but also for physicists. Since the Belousov–Zhabotinskii reaction pattern, a well‐known non‐linear phenomenon in chemistry, was observed during aggregation of Dictyostelium amoebae, Dictyostelium has been one of the major subjects of non‐linear dynamics studies. Macroscopic theory, such as continuous cell density approximation, has been a common approach to studying pattern formation since the pioneering work of Turing. Recently, promising microscopic approaches, such as the cellular dynamics method, have emerged. They have shown that Dictyostelium is useful as a model system in biology. The synchronization mechanism of oscillatory production of cyclic adenosine 3′,5′‐monophosphate in Dictyostelium is discussed in detail to show how it is a universal feature that can explain synchronization in other organisms.
Change in the adhesive properties of blastomeres during early cleavage stages in sea urchin embryo
Authors: Masui Mizuko , Kominami Tetsuya
Published: March 2003

Blastomeres of sea urchin embryo change their shape from spherical to columnar during the early cleavage stage. It is suspected that this cell shape change might be caused by the increase in the adhesiveness between blastomeres. By cell electrophoresis, it was found that the amount of negative cell surface charges decreased during the early cleavage stages, especially from the 32‐cell stage. It was also found that blastomeres formed lobopodium‐like protrusions if the embryos were dissociated in the presence of Ca2+. Interestingly, a decrease in negative cell surface charges and pseudopodia formation first occurred in the descendants of micromeres and then in mesomeres, and last in macromeres. By examining the morphology of cell aggregates derived from the isolated blastomeres of the 8‐cell stage embryo, it was found that blastomeres derived from the animal hemisphere (mesomere lineage) increased their adhesiveness one cell cycle earlier than those of the vegetal hemisphere (macromere lineage). The timing of the initiation of close cell contact in the descendants of micro‐, meso‐ and macromeres was estimated to be 16‐, 32‐ and 60‐cell stage, respectively. Conversely, the nucleus‐to‐cell‐volume ratios, which are calculated from the diameters of the nucleus and cell, were about 0.1 when blastomeres became adhesive, irrespective of the lineage.
Localization of mitochondrial ribosomal RNA on the chromatoid bodies of marine planarian polyclad embryos
Authors: Sato Kimihiro , Sugita Tomoe , Kobayashi Kazuya , Fujita Kaoru , Fujii Takeshi , Matsumoto Yohei , Mikami Tetsuya , Nishizuka Natsue , Nishizuka Sanae , Shojima Keisuke , Suda Miho , Takahashi Gen , Himeno Hyouta , Muto Akira , Ishida Sachiko
Published: March 2003

Electron‐dense cytoplasmic structures, referred to as chromatoid bodies, are observed in the somatic stem cells, called neoblasts, and germline cells in adult planarians. Although it has been revealed that the chromatoid bodies morphologically resemble germline granules in Drosophila and Xenopus embryos, what essential role it plays in the planarian has remained unclear. In the present study, to examine whether chromatoid bodies in planarian embryos are responsible for germline formation, the presence and behavior of chromatoid bodies during embryogenesis were examined. Mitochondrial large ribosomal RNA and mitochondrial small ribosomal RNA were used as candidate markers for components of the chromatoid body. Starting from the fertilized egg, extramitochondrial signals of both RNA (mtrRNA) were observed. At the ultrastructural level, mtrRNA were localized on the surface of the chromatoid bodies. At subsequent stages, the signals of mtrRNA were observed in certain restricted blastomeres that contribute to the formation of larval structures. The signals gradually decreased from the gastrula stage. These results suggest that the chromatoid bodies associated with mtrRNA in embryogenesis are not germline granules. The chromatoid bodies of blastomeres may be concerned with the toti‐ or pluripotency and cell differentiation as proposed in adult planarian neoblasts.
Cell proliferation and protein synthesis as initial factors in determination of axial polarity
Authors: Flickinger R. A.
Published: March 2003

The rate of cell proliferation relative to that of protein synthesis appears to have an initial role in establishment of axial polarities in developing animal embryos. An increase in this ratio leads to anterior or dorsal differentiation, while reduction allows posterior or ventral differentiation in a number of organisms. The role that various growth factors play in the regulation of proliferation and protein synthesis is examined.
Myb‐binding site regulates the expression of glucosamine‐6‐phosphate isomerase in Dictyostelium discoideum
Authors: Tabata Kazuhito , Matsuda Yuki , Viller Eric , Masamune Yukito , Katayama Takahiro , Yasukawa Hiroo
Published: March 2003

A homolog of the glucosamine‐6‐phosphate isomerase in the cellular slime mold Dictyostelium discoideum has been analyzed. The gene disruption mutant was arrested at the mound stage, demonstrating that the gene is important for development. The gene was expressed in vegetatively growing cells, silenced on starvation and expressed again in prestalk cells during the multicellular stages. The upstream region of the gene (1376 bp relative to ATG) was cloned and sequenced to study the transcription control mechanisms. Analysis of deletion mutants and a site‐directed mutant indicated that the Myb‐binding sequence (5′‐AACTG‐3′) localized in the upstream region is important for gene expression. The results of gel‐shift assays showed the presence of an Myb‐related protein binding to the sequence at the growing phase and another protein binding to the sequence at developmental stages.
Presence of a nucleus or nucleus‐deriving factors is indispensable for the formation of the spindle, the diastema and the cleavage furrow in the blastomere of the Xenopus embryo
Authors: Wakabayashi Yasunobu , Shinagawa Atsunori
Published: March 2003

The present study examines the indispensability of a nucleus or nucleus‐deriving factors in the induction of cleavage in Xenopus eggs by testing cleavage in Xenopus eggs fertilized with ultraviolet (UV)‐damaged sperm and deprived of the female nucleus. These eggs, which contain only one UV‐damaged nucleus with one set of centrioles, undergo unique cleavages. Cleavage takes place in only one of the two blastomeres formed by the immediately preceding cleavage. Histologically, only one nucleus, which does not appear to be organized into typical chromosomes, is found in one of the two blastomeres formed by the immediately preceding cleavage. The typical bipolar spindle and the diastema, or a slit of astral rays, are formed in the blastomere that contains the nucleus. By contrast, only asters lacking the spindle and the diastema are formed in the remaining blastomeres, which do not contain a nucleus. The same results are obtained in eggs that contain two UV‐damaged nuclei with one set of centrioles. In these eggs, cleavage appears to occur in one or two blastomeres that contain either or both of the nuclei and one bipolar spindle. In eggs that contain one intact and one UV‐damaged nuclei, cleavage takes place quite normally with each blastomere containing one nucleus or one set of chromosomes as well as one bipolar spindle. Thus, there is a very close correlation between the presence of a nucleus and the formation of the mitotic spindle, the diastema and the cleavage furrow in the blastomeres of Xenopus embryos. We conclude that the presence of a nucleus or nucleus‐deriving factors is indispensable for the formation of the bipolar spindle, the diastema and the cleavage furrow in the blastomeres of the Xenopus embryos..
Process of pigment cell specification in the sand dollar, Scaphechinus mirabilis
Authors: Kominami Tetsuya , Takata Hiromi
Published: March 2003

The process of pigment cell specification in the sand dollar Scaphechinus mirabilis was examined by manipulative methods. In half embryos, which were formed by dissociating embryos at the 2‐cell stage, the number of pigment cells was significantly greater than half the number of pigment cells observed in control embryos. This relative increase might have been brought about by the change in the arrangement of blastomeres surrounding the micromere progeny. To examine whether such an increase could be induced at a later stage, embryos were bisected with a glass needle. When embryos were bisected before 7 h postfertilization, the sum of pigment cells observed in a pair of embryo fragments was greater than that in control embryos. This relative increase was not seen when embryos were bisected after 7 h postfertilization. From the size of blastomeres, it became clear that the 9th cleavage was completed by 7 h postfertilization. Aphidicolin treatment revealed that 10–15 pigment founder cells were formed. The results obtained suggest that the pigment founder cells were specified through direct cell contact with micromere progeny after the 9th cleavage, and that most of the founder cells had divided three times before they differentiated into pigment cells.
Study of Cynops pyrrhogaster notochord differentiation using a novel monoclonal antibody
Authors: Suzuki Akio S. , Sakaguchi Koji , Tajima Tatsuya , Sasaki Takumi , Imoh Hiroshi
Published: March 2003

Two monoclonal antibodies which reacted specifically with the notochord of the early Cynops pyrrhogaster embryo were screened. The antigen molecules were detected within and around the notochord. They were first found mostly between the neural plate and the dorsal part of the notochord in the early neurula (stage 15). They were subsequently detected between the notochord and the somite in the advanced embryo, and they were last detected between the notochord and the underlying endoderm. Whole‐mount labeling indicated that the antigen molecules were first detected in the anterior half of the notochord in the early neurula (stage 15). The signals gradually spread along the anterior–posterior axis, especially towards the posterior region. This fact suggests that notochord differentiation progresses from the anterior region which first receives the dorsal mesoderm‐inducing signals released horizontally from the lower dorsal marginal zone during early gastrulation. The present study suggested that: (i) notochord differentiation proceeds from the anterior region; and (ii) secretion of the antigen molecules results in the drawing of a boundary between the adjacent tissues.
Tissue‐specific regulation of type III iodothyronine 5‐deiodinase gene expression mediates the effects of prolactin and growth hormone in Xenopus metamorphosis
Authors: Shintani Noriyuki , Nohira Takashi , Hikosaka Akira , Kawahara Akira
Published: March 2003

Prolactin (PRL) and growth hormone (GH) are known to be able to act as antimetamorphic hormones. From investigations of how PRL inhibits Xenopus tail regression in vitro, it was found that the both hormones could, in addition to their known antimetamorphic actions, upregulate mRNA expression of type III iodothyronine 5‐deiodinase (5D), an enzyme that inactivates thyroid hormones (TH). Conversely, both PRL and GH were found to downregulate 5D mRNA expression in the liver. Blockage by PRL of TH‐induced tail regression in organ culture was released by treatment with iopanoic acid (IOP, an inhibitor of 5D activity). The IOP‐released tail regression displayed a unique morphology of the larger fins retained on the regressing tails, consistent with the finding that mRNA for both PRL receptor and 5D were enriched in the fin. The results suggest that the metamorphosis‐modulating actions of PRL and GH are mediated, at least partially, by tissue‐specific regulation of 5D mRNA expression.
Extrachromosomal transposition of the transposable element Minos in embryos of the cricket Gryllus bimaculatus
Authors: Zhang Hongjie , Shinmyo Yohei , Hirose Ayumi , Mito Taro , Inoue Yoshiko , Ohuchi Hideyo , Loukeris Thanasis G. , Eggleston Paul , Noji Sumihare
Published: March 2003

Effective germline transformation of insects has been shown to depend on the right choice of transposon system and selection marker. In this study the promoter region of a Gryllus cytoplasmic actin (GbA3/4) gene was isolated and characterized, and was used to drive the expression of Minos transposase in embryos of the cricket Gryllus bimaculatus. Active Minos transposase was produced in these embryos as monitored through established transposon excision and interplasmid transposition assays. In contrast, Drosophila melanogaster hsp70 promoter, previously used to express Minos transposase in a number of insect species and insect cell lines, failed to produce any detectable Minos transposase activity, as recorded by using the very sensitive transposon excision assay. In addition, the GbA3/4 promoter was found to drive expression of enhanced green fluorescent protein (eGFP) predominantly in vitellophages of the developing Gryllus eggs when a plasmid carrying a GbA3/4 promoter‐eGFP fusion gene was transiently injected into embryos. These results strongly support the use of Minos transposons marked with the GbA3/4 promoter‐eGFP for the genetic transformation of this emerging model insect species.
Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: Partial characterization and implication in metamorphosis
Authors: Izutsu Yumi , Tochinai Shin , Maéno Mitsugu , Iwabuchi Kazuya , Onoé Kazunori
Published: March 2003

It has been shown that larval skin (LS) grafts are rejected by an inbred strain of adult Xenopus, which suggests a mechanism of metamorphosis by which larval cells are recognized and attacked by the newly differentiating immune system, including T lymphocytes. In an attempt to define the larval antigenic molecules that are targeted by the adult immune system, anti‐LS antibodies (IgY) were produced by immunizing adult frogs with syngeneic LS grafts. The antigen molecules that reacted specifically with this anti‐LS antiserum were localized only in the larval epidermal cells. Of 53 and 59–60 kDa acidic proteins that were reactive with anti‐LS antibodies, a protein of 59 kDa and with an isoelectric point of 4.5 was selected for determination of a 19 amino acid sequence (larval peptide). The rat antiserum raised against this peptide was specifically reactive with the 59 kDa molecules of LS lysates. Immunofluorescence studies using these antisera revealed that the larval‐specific molecules were localized in both the tail and trunk epidermis of premetamorphic larvae, but were reduced in the trunk regions during metamorphosis, and at the climax stage of metamorphosis were detected only in the regressing tail epidermis. Culture of splenocytes from LS‐immunized adult frogs in the presence of larval peptide induced augmented proliferative responses. Cultures of larval tail pieces in T cell‐enriched splenocytes from normal frogs or in natural killer (NK)‐cell‐enriched splenocytes from early thymectomized frogs both resulted in significant destruction of tail pieces. Tissue destruction in the latter was enhanced when anti‐LS antiserum was added to the culture. These results indicate that degeneration of tail tissues during metamorphosis is induced by a mechanism such that the larval‐specific antigen molecules expressed in the tail epidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.
Contribution of micro‐organisms to the carbon dynamics in black spruce (Picea mariana) forest soil in Canada
Authors: Uchida Masaki , Nakatsubo Takayuki , Horikoshi Takao , Nakane Kaneyuki
Published: March 2003

In order to clarify the role of micro‐organisms in the carbon cycle of the boreal forest ecosystem, the vertical distribution of soil carbon, soil microbial biomass and respiratory activity was studied in a black spruce forest near Candle Lake in Saskatchewan, Canada. The total amount of carbon contained in moss and soil layers (to the depth of 50 cm beneath the mineral soil surface) was 7.2 kg m−2, about 47% of which was in the L and FH horizons of the soil. Soil microbial biomass per dry weight of soil was largest in the L horizon, while the biomass per ground area was largest in the FH horizon. Soil respiration rate, measured using a portable infrared gas analyzer, was highest in the FH horizon, exceeding 50% of the total soil respiration. Low but significant CO2 emission was detected even in deeper soil horizon (E horizon). We also examined the respiration rate of cut roots and the effect of root excision on respiration. The contribution of root respiration to total soil respiration, calculated from root biomass and respiration rate of cut roots, was about 54%. The amount of carbon evolved through microbial respiration during the snow‐free season ( June–October) was estimated as 221 g C m−2. Micro‐organisms in the L horizon showed high respiratory activity as compared with those in deeper soil horizons.
The role of sexual and clonal reproduction in maintaining population inFritillaria camtschatcensis(L.) Ker‐Gawl. (Liliaceae)
Authors: Shimizu Takaaki , Hatanaka Yasuo , Zentoh Hiroko , Yashima Takeshi , Kinoshita Eiichiro , Watano Yasuyuki , Shimizu Tatemi
Published: March 2003

Fritillaria camtschatcensiscan reproduce by means of both sexual reproduction and clonal multiplication. Despite prolific seed production, no seedlings have been found for several years in natural populations on Mt Hakusan. The purposes of this paper are to clarify: (i) whether population size is maintained mainly by clonal multiplication; and (ii) if this is the case, to what extent occasional seedling establishment affects population growth rate and population structure. Two permanent quadrats were placed in subalpine meadows in 1992 on Mt Hakusan. Plant size, location and reproductive states for all ramets in the quadrats were recorded every year. Projection matrices were created based on field census, and computer simulation experiments were performed.Fritillaria camtschatcensishad two types of flower, male flower and cosexual flower, and they were changeable. This is the first report on sex lability inFritillaria. Clonal growth was more closely correlated with life‐history stages, especially with sexual states than with plant size. The population growth rate,λ, was 1.006 for the Mizuyajiri population and 1.047 for the Nanryu population, respectively. Seedlings were found in 1996 for the first time. These facts indicate that populations ofF. camtschatcensison Mt Hakusan can usually be maintained by clonal multiplication. However, it is not yet certain whether seeds germinate every year or whether a flush of seedling emergence occurs once in every few years in natural populations. Computer simulation revealed that: (i) there was a critical germination rate above which population growth rate suddenly increased; and (ii) occasional seedling establishment could provide almost the same contribution to population growth rate as that of annual seedling establishment. These results suggest that population size can be maintained mainly by clonal multiplication, and the role of sexual reproduction lies beyond maintaining the population size inF. camtschatcensis.
Pruning behavior and intercolony competition ofTetraponera (Pachysima) aethiops(Pseudomyrmecinae, Hymenoptera) inBarteria fistulosain a tropical forest, Democratic Republic of Congo
Authors: Yumoto Takakazu , Maruhashi Tamaki
Published: March 2003

The early stages of colonization byTetraponera(=Pachysima)aethiops(Pseudomyrmecinae) to its obligate host plant,Barteria fistulosa(Passifloraceae), was studied in Kahuzi‐Biega National Park, Democratic Republic of Congo (formerly Zaire). In our observations, as many as 36 queens colonized a seedling, and all of the established colonies had just a single queen each. The ants depend totally on the products of scale insects which inhabit the domatia. The ants damage the terminal meristems of the branches where other incipient colonies may inhabit. This tip‐biting behavior may deteriorate the nutritional condition of scale insects and suppress the growth of the competing colonies. Ants pruned the plants near the edge of the trunk of the host tree most intensively, even the foliage of the lowest branch in shade. This observation supports the hypothesis that the main driving force which leads to pruning behavior is intercolony competition rather than improving the light condition of the host tree.
Effects of bird ingestion on seed germination of two Prunus species with different fruit‐ripening seasons
Authors: YAGIHASHI TSUTOMU , HAYASHIDA MITSUHIRO , MIYAMOTO TOSHIZUMI
Published: March 2003

To evaluate the effects of ingestion by birds on seed germination under natural conditions, we carried out germination experiments in the field using seeds of two Prunus species that have different fruit‐ripening seasons. Germination of seeds with the following three treatments was compared: ‘ingested seeds’, seeds excreted after feeding of fruits to birds; ‘extracted seeds’, seeds deliberately extracted from the fruit pulp; and ‘intact fruit’, seeds in untreated intact fruit. Many ingested and extracted seeds of both Prunus species germinated during the first spring, and the difference in germination percentage between ingested and extracted seeds was not significant. Many seeds in intact fruit of Prunus sargentii also germinated during the first spring, but those of Prunus ssiori did not germinate until the second spring. Pulp removal through bird ingestion enabled rapid germination for the autumn‐fruiting P. ssiori, whose fruit pulp was not likely to be decomposed until the first spring. In contrast, the effects of ingestion were not striking for the summer‐fruiting P. sargentii, whose fruit pulp is quickly decomposed.
Early stages of secondary succession on abandoned cropland in north‐east Borneo Island
Authors: Ohtsuka Toshiyuki
Published: March 2003

A study on community structure and floristic change in the early stages of the tropical old‐field succession in an intensive shifting cultivation area, Sabah, north‐east Borneo Island, was conducted in March and September 1997. Dense therophytic communities were established soon after abandonment, and rapidly changed: namely, from Galinsoga parviflora (in approximately 1–2 months) to Crassocephalum crepidioides (in approximately 3–5 months) and further to Conyza sumatrensis (in approximately 1 year). These therophytic communities were replaced by perennial grass and shrub species, such as Imperata cylindrica and Eupatorium odoratum, within 3 years after abandonment. At this stage, a few pioneer tree species such as Trema orientalis made a sparse canopy layer approximately 3–4 m high. The three pioneer annuals have wind‐dispersed small seeds. The plant densities of the three annuals did not appear to change between areas fallow for 1 month and 4 months. Almost all of the seedlings of the three annuals might simultaneously have invaded the fallowed field soon after abandonment. The three pioneer annuals take a different amount of time to reach their respective mature sizes. This resulted in the rapid successional changes in the tropical old‐field succession. The seed‐bank annuals, whose seeds germinate after the winter pre‐chilling, occurred in the temperate old‐fields as the first‐year pioneers under seasonal condition. Intermittent shifting cultivation and tropical conditions without seasonality favour the occurrence of wind‐dispersed annuals with a spatially fugitive strategy.
Floaters as intraspecific brood parasites in the grey starling Sturnus cineraceus
Authors: Saitou Takashi
Published: March 2003

The breeding season of the grey starling Sturnus cineraceus is divided into two periods: the early and late breeding season. The birds that breed in each season are referred to as early and late breeders, respectively. In this study, the late breeders mainly consisted of new immigrants that did not breed in the early season. This suggested that these new immigrants were probably floaters in the early breeding season. Because intraspecific brood parasitism occurred frequently, it is possible that the parasites were floaters without nesting boxes. To check for the presence of floaters, two field experiments were conducted and floaters were captured with traps. With additional nesting boxes provided during the breeding season, all new boxes were quickly occupied by floaters from the period of incubation to hatching in the early breeding season, but were not occupied by floaters during the egg laying period of the late breeding season. The addition of boxes before the start of the breeding season significantly decreased the parasitic rate and number of parasitic eggs per nest. There was a positive correlation between the relative occupancy of nesting boxes and the parasitic rate. The removal of boxes again increased the parasitic rate. As for the capture of floaters with traps, the number of trapped birds per day was also related to the relative occupancy of nesting boxes. The floaters trapped between incubation and nesting periods of early breeders became the late breeders. Judging from these results, many floaters were present from the incubation to hatching periods of early breeders, and were probably intraspecific brood parasites.
Growth characteristics of six tree species on Hokkaido Island, northern Japan
Authors: Umeki Kiyoshi
Published: March 2003

Based on growth data obtained from 13 804 individual trees in 110 permanent plots, diameter at breast height (d.b.h.) growth was modeled for six major tree species growing in Hokkaido Island, northern Japan. For each species, d.b.h. growth was regressed on independent variables, indicating the effects of initial size, competition, climate and topography. In developing the d.b.h. growth models, independent variables were selected using the stepwise method. For all species analyzed, the final models accounted for significant amounts of the variation (r2= 5.65−20.99%;P< 5%) in d.b.h. growth. Significant (P< 5%) effects of competition, climate and topography on d.b.h. growth were detected. The model obtained can be used in simulations of forest dynamics. Some ecological questions were examined using the d.b.h. growth models obtained. For hardwoods, the d.b.h. growth rate did not correlate with the published grouping of species in terms of shade‐tolerance; however, the plasticity in d.b.h. growth in the presence of competition seemed to be correlated with the shade‐tolerance of species. The degree of asymmetry in competition evaluated for each species was not correlated with the availability of underground resources, which was expressed by surrogate variables (mean values of climatic and topographic variables for species). The degree of asymmetry in competition was not different between conifers and hardwoods.
Gap formation, microsite variation and the conifer seedling occurrence in a subalpine old‐growth forest, central Japan
Authors: Narukawa Yoshiko , Yamamoto Shin‐Ichi
Published: March 2003

To evaluate the effects of canopy gaps and forest floor microsites (soil, fallen logs, root‐mounds, buttresses and stumps) on regeneration of subalpine forests, the gap regeneration and seedling occurrence of conifers (Abies mariesii, Abies veitchii, Picea jezoensis var. hondoensis and Tsuga diversifolia) were studied in two stands of a subalpine old‐growth forest, central Japan. The percentage of gap area to total surveyed area was 11.2–11.3% in the stands. Gap regeneration was not common for P. jezoensis var. hondoensis and T. diversifolia. In contrast, gap regeneration by advanced regeneration was relatively common for Abies. Seedling occurrence of P. jezoensis var. hondoensis and T. diversifolia was restricted on elevated surfaces such as stumps and root‐mounds, while Abies seedlings could occur on soil as well as on elevated surfaces. Rotten stumps were the most favorable microsites for conifer seedling occurrence, which covered small area in the forest floor. Although canopy gaps were not always favorable for seedling occurrence, all conifer seedlings were larger under canopy gaps than under closed canopy. Canopy gaps and forest floor microsites clearly affected seedling occurrence and growth of conifers. This suggests that regeneration of conifers is related to the difference of growth advantage under canopy gaps and favorable microsites for seedling occurrence.
Cospeciation of ants and plants
Authors: Itino Takao , Davies Stuart J. , Tada Hideko , Hieda Yoshihiro , Inoguchi Mika , Itioka Takao , Yamane Seiki , Inoue Tamiji
Published: March 2003

Cospeciation, in which both parties of an ecological interaction speciate in parallel with each other, has rarely been reported in biotic associations except the cases for host–parasite interaction. Many tropical plants house ants and thereby gain protection against herbivores. Although these ant–plant symbioses have been regarded as classical cases of coevolved mutualism, no evidence of cospeciation has been documented. The Asian ant–plant association betweenCrematogasterants andMacarangaplants is highly species specific and the molecular phylogeny of the ants parallels the plant phylogeny, reflecting history of cospeciation. Evidence is presented that this association has been maintained over the past seven million years. Phylogeographic patterns of 27 ants from twoMacarangaspecies suggest that allopatric cospeciations are still in progress in Asian wet tropics.