bluuebunny/crossref_metadata_2025 huggingface dataset:
An evolutionarily conserved tripartite tryptophan motif stabilizes the prodomains of cathepsin L‐like cysteine proteases
Authors: Kreusch Stefan , Fehn Mark , Maubach Gunter , Nissler Karl , Rommerskirch Winfried , Schilling Klaus , Weber Ekkehard , Wenz Ingrid , Wiederanders Bernd
Published: October 2003

Cathepsin L‐like cysteine proteinases contain an evolutionarily highly conserved α‐helical motif in the proregion. This is called the ER(F/W)N(I/V)N motif according to the conserved amino acids along one side of the helix. We studied the function of this motif using site‐directed mutagenesis experiments of human procathepsin S. We replaced each of these amino acids with alanine and constructed deletion mutants lacking parts of the helix. All mutants were expressed in HEK 293 cells, but only one, W52A, was not processed to mature cathepsin S, nor was it phosphorylated or secreted into the culture medium. W52 is part of the hydrophobic core in the propeptide region of cathepsin S comprising two additional tryptophan residues, W28 and W31, also conserved among cathepsin L‐like cysteine peptidases. Replacement of the latter with alanine led to consequences similar to those with the W52A mutation. Recombinant propeptides containing mutations of one of the three tryptophan residues were three orders of magnitude less effective as inhibitors of mature cathepsin S than the wild‐type propeptide. The results point to a dominant role of the respective hydrophobic stack in the proper folding, transport and maturation of procathepsin S and related cathepsin L‐like cysteine proteinases.
Binding of rat brain hexokinase to recombinant yeast mitochondria
Authors: Azoulay‐Zohar Heftsi , Aflalo Claude
Published: October 2003

The association of rat brain hexokinase with heterologous recombinant yeast mitochondria harboring human porin (Yh) is comparable to that with rat liver mitochondria in terms of cation requirements, cooperativity in binding, and the effect of amphipathic compounds. Mg2+, which is required for hexokinase binding to all mitochondria, can be replaced by other cations. The efficiency of hexokinases, however, depends on the valence of hydrophilic cations, or the partition of hydrophobic cations in the membrane, implying that these act by reducing a prohibitive negative surface charge density on the outer membrane rather than fulfilling a specific structural requirement. Macromolecular crowding (using dextran) has dual effects. Dextran added in excess increases hexokinase binding to yeast mitochondria, according to the porin molecule they harbor. This effect, significant with wild‐type yeast mitochondria, is only marginal with Yh as well as rat mitochondria. On the other hand, an increase in the number of hexokinase binding sites on mitochondria is also observed. This increase, moderate in wild‐type organelles, is more pronounced with Yh. Finally, dextran, which has no effect on the modulation of hexokinase binding by cations, abolishes the inhibitory effect of amphipathic compounds. Thus, while hexokinase binding to mitochondria is predetermined by the porin molecule, the organization of the latter in the membrane plays a critical role as well, indicative that porin must associate with other mitochondrial components to form competent binding sites on the outer membrane.
Identification, purification and characterization of an acetoacetyl‐CoA thiolase from rat liver peroxisomes
Authors: Antonenkov Vasily D. , Croes Kathleen , Waelkens Etienne , Van Veldhoven Paul P. , Mannaerts Guy P.
Published: October 2003

Acetoacetyl‐CoA specific thiolases catalyse the cleavage of acetoacetyl‐CoA into two molecules of acetyl‐CoA and the synthesis (reverse reaction) of acetoacetyl‐CoA. The formation of acetoacetyl‐CoA is the first step in cholesterol and ketone body synthesis. In this report we describe the identification of a novel acetoacetyl‐CoA thiolase and its purification from isolated rat liver peroxisomes by column chromatography. The enzyme, which is a homotetramer with a subunit molecular mass of 42 kDa, could be distinguished from the cytosolic and mitochondrial acetoacetyl‐CoA thiolases by its chromatographic behaviour, kinetic characteristics and partial internal amino‐acid sequences. The enzyme did not catalyse the cleavage of medium or long chain 3‐oxoacyl‐CoAs. The enzyme cross‐reacted with polyclonal antibodies raised against cytosolic acetoacetyl‐CoA thiolase. The latter property was exploited to confirm the peroxisomal localization of the novel thiolase in subcellular fractionation experiments.

 The peroxisomal acetoacetyl‐CoA thiolase most probably catalyses the first reaction in peroxisomal cholesterol and dolichol synthesis. In addition, its presence in peroxisomes along with the other enzymes of the ketogenic pathway indicates that the ketogenic potential of peroxisomes needs to be re‐evaluated.
Cloning, overexpression and mutagenesis of cDNA encoding dihydrolipoamide succinyltransferase component of the porcine 2‐oxoglutarate dehydrogenase complex
Authors: Koike Kichiko , Suematsu Takashi , Ehara Masahiko
Published: October 2003

Dihydrolipoamide succinyltransferase (E2o) is the structural and catalytic core of the 2‐oxoglutarate dehydrogenase (OGDH) complex. The cDNA encoding porcine E2o (PE2o) has been cloned. The PE2o cDNA spans 2547 bases encoding a presequence (68 amino‐acid residues) and a mature protein (387 residues, Mr = 41 534). Recombinant porcine E2o (rPE2o) (residues 1–387), C‐ and N‐terminal truncated PE2os, and site‐directed mutant PE2os were overexpressed in Escherichia coli via the expression vector pET‐11d and purified. The succinyltransferase activity of the rPE2o was about 2.2‐fold higher than that of the native PE2o. Electron micrographs of the rPE2o negatively stained showed a cube‐like structure very similar to that of the native PE2o. Deletion of five amino‐acid residues from the C‐terminus resulted in a complete loss of both enzymatic activity and formation of the cube‐like structure, but the deletion of only the last two residues had no effect on either function, suggesting the important roles of the C‐terminal leucine triplet (Leu383–384–385). Substitution of Ser306 with Ala, and Asp362 with Asn, Glu or Ala in the putative active site, and Leu383–384–385 with Ala or Asp abolished both functions. Substitution of His358 with Cys resulted in an 8.5‐fold reduction in kcat, with little change in Km values for dihydrolipoamide and succinyl‐CoA. However, self‐assembly was not affected. These data indicate that Ser306, Asp362 and the Leu383–384–385 triplet are important residues in both the self‐assembly and catalytic mechanism of PE2o.
Novel high‐affinity photoactivatable antagonists of corticotropin‐releasing factor (CRF)
Authors: Bonk Ines , Rühmann Andreas
Published: October 2003

Novel photoactivatable antagonists of human/rat corticotropin‐releasing factor (h/rCRF) have been synthesized and characterized. The N‐terminal amino acid d‐phenylalanine in astressin {cyclo(30–33) [d‐Phe12, Nle21,38, Glu30, Lys33]h/rCRF‐(12–41)}, a potent CRF peptide antagonist, was replaced by a phenyldiazirine, the 4‐(1‐azi‐2,2,2‐trifluoroethyl)benzoyl (ATB) residue. Additionally, His32 of astressin was substituted by either alanine or tyrosine for specific radioactive labeling with 125I at either His13 or Tyr32, respectively. The photoactivatable CRF antagonists were tested for their ability to displace 125I‐labeled Tyr0 ovine CRF ([125I‐labeled Tyr0]oCRF) in binding experiments and to inhibit oCRF‐stimulated adenylate cyclase activity in human embryonic kidney (HEK) 293 cells, permanently transfected with cDNA coding for rat CRF receptor, type 1 (rCRFR1) or human Y‐79 retinoblastoma cells known to carry endogenous functional human CRFR1 (hCRFR1). ATB‐cyclo(30–33)[Nle21,38, Glu30, Ala32, Lys33]h/rCRF‐(13–41) (compound 1) was found to bind with higher affinity to rat or human CRFR1 when compared with ATB‐cyclo(30–33)[Nle21,38, Glu30, Tyr32, Lys33]h/rCRF‐(13–41) (compound 2) and exhibited higher inhibition of oCRF‐stimulated cAMP accumulation in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK‐rCRFR1 cells) or Y‐79 cells. A highly glycosylated, 66‐kDa protein was identified with SDS/PAGE, when the radioactively iodinated compounds 1 or 2 were covalently linked to rCRFR1. The specificity of the photoactivatable 125I‐labeled CRF antagonists was demonstrated with SDS/PAGE by the finding that these analogs could be displaced from the receptor by their corresponding nonlabeled form, but not other unrelated peptides such as vasoactive intestinal peptide. The observed molecular size of the receptor was in agreement with the size of CRFR1 found in rat pituitary (66 kDa), but was significantly larger than the size of CRFR1 found in rat cerebellum and olfactory bulb (53 kDa).
Effects of aluminum on activity of Krebs cycle enzymes and glutamate dehydrogenase in rat brain homogenate
Authors: Zatta P. , Lain E. , Cagnolini C.
Published: October 2003

Aluminum is a neurotoxic agent for animals and humans that has been implicated as an etiological factor in several neurodegenerative diseases and as a destabilizer of cell membranes. Due to its high reactivity, Al3+ is able to interfere with several biological functions, including enzymatic activities in key metabolic pathways. In this paper we report that, among the enzymes that constitute the Krebs cycle, only two are activated by aluminum: α‐ketoglutarate dehydrogenase and succinate dehydrogenase. In contrast, aconitase, shows decreased activity in the presence of the metal ion. Al3+ also inhibits glutamate dehydrogenase, an allosteric enzyme that is closely linked to the Krebs cycle. A possible correlation between aluminum, the Krebs cycle and aging processes is discussed.
Probing ligand‐induced conformational changes of human CD38
Authors: Berthelier Valérie , Laboureau Julien , Boulla Geneviève , Schuber Francis , Deterre Philippe
Published: October 2003

The lymphoid surface antigen CD38 is basically a NAD+glycohydrolase, which is also involved in the metabolism of cyclic ADP‐ribose. Besides, this ecto‐enzyme has potential signalling roles in T‐ and B‐cells. Such multiple functions prompted us to study the molecular dynamics of the CD38 protein and especially the relationship between its ecto‐enzymatic active site and its epitope, i.e. the binding site of most known anti‐CD38 monoclonal antibodies. Both epitopic and enzymatic sites were shown to be degraded by proteases, such as trypsin or chymotrypsin. This sensitivity was almost entirely suppressed in the presence of substrates or inhibitors. Both sites were also degraded in the presence of reducing agents, as dithiothreitol. Inhibitory ligands induced the same resistance of both sites against reducing attack. The binding of CD38 ligands to the active site triggers therefore conformational changes that shield some backbone bonds and disulfide bridges against, respectively, proteolytic cleavage or reduction. This transconformation was found moreover to irreversibly take place after incubation with substrates such as NAD+ in the presence of dithiothreitol. The epitope remained preserved, while the enzymatic activity was lost. This inactivation probably resulted from the covalent trapping of the catalytically reactive intermediate in the active site (i.e. paracatalytic inactivation). These data have major implications in the knowledge of the CD38 structure, especially with regard to the location of disulfide bridges and their accessibility. Potential consequences of the conformational plasticity of CD38 should also be considered in its physiological functions such as signalling.
Interaction between the lipoamide‐containing H‐protein and the lipoamide dehydrogenase (L‐protein) of the glycine decarboxylase multienzyme system
Authors: Faure Magali , Bourguignon Jacques , Neuburger Michel , Macherel David , Sieker Larry , Ober Raymond , Kahn Richard , Cohen‐Addad Claudine , Douce Roland
Published: October 2003

The glycine decarboxylase complex consists of four different component enzymes (P‐, H‐, T‐ and L‐proteins). The 14‐kDa lipoamide‐containing H‐protein plays a pivotal role in the complete sequence of reactions as its prosthetic group (lipoic acid) interacts successively with the three other components of the complex and undergoes a cycle of reductive methylamination, methylamine transfer and electron transfer. With the aim to understand the interaction between the H‐protein and its different partners, we have previously determined the crystal structure of the oxidized and methylaminated forms of the H‐protein. In the present study, we have crystallized the H‐protein in its reduced state and the L‐protein (lipoamide dehydrogenase or dihydrolipoamide dehydrogenase). The L‐protein has been overexpressed in Escherichia coli and refolded from inclusion bodies in an active form. Crystals were obtained from the refolded L‐protein and the structure has been determined by X‐ray crystallography. This first crystal structure of a plant dihydrolipoamide dehydrogenase is similar to other known dihydrolipoamide dehydrogenase structures. The crystal structure of the H‐protein in its reduced form has been determined and compared to the structure of the other forms of the protein. It is isomorphous to the structure of the oxidized form. In contrast with methylaminated H‐protein where the loaded lipoamide arm was locked into a cavity of the protein, the reduced lipoamide arm appeared freely exposed to the solvent. Such a freedom is required to allow its targeting inside the hollow active site of L‐protein. Our results strongly suggest that a direct interaction between the H‐ and L‐proteins is not necessary for the reoxidation of the reduced lipoamide arm bound to the H‐protein. This hypothesis is supported by biochemical data [Neuburger, M., Polidori, A_m., Piètre, E., Faure, M., Jourdain, A., Bourguignon, J., Pucci, B. & Douce, R. (2000) Eur. J. Biochem.267, 2882–2889] and by small angle X‐ray scattering experiments reported herein.
Germ cell‐specific heat shock protein 105 binds to p53 in a temperature‐sensitive manner in rat testis
Authors: Kumagai Jin , Fukuda Jun , Kodama Hideya , Murata Masanori , Kawamura Kazuhiro , Itoh Hideaki , Tanaka Toshinobu
Published: October 2003

Heat shock protein (HSP)105 is a testis‐specific and HSP90‐related protein. The aim of this study was to explore the functions of HSP105 in the rat testis. Signals of HSP105 were detected immunohistochemically in the germ cells and translocated from the cytoplasm to the nucleus at 2 days after experimental induction of cryptorchidism. In cultured testicular germ cells, a significant increase in the expression of HSP105 in response to heat stress (37 °C) was detected in the insoluble protein fractions. Several binding proteins were isolated from rat testis using a HSP105 antibody immunoaffinity column, and p53, the tumor suppressor gene product, was copurified with these. Furthermore, immunoprecipitation using antibodies to p53 led to coprecipitation of HSP105 together with p53 after culturing germ cells at 32.5 °C, but not at 37 or 42 °C. In conclusion, HSP105 is specifically localized in the germ cells and may translocate into the nucleus after heat shock. HSP105 is suggested to form a complex with p53 at the scrotal temperature, and dissociate from it at suprascrotal temperatures. At scrotal temperature, HSP105 may thus contribute to the stabilization of p53 proteins in the cytoplasm of the germ cells, preventing the potential induction of apoptosis by p53.
Functional expression of mung bean Ca2+/H+ antiporter in yeast and its intracellular localization in the hypocotyl and tobacco cells
Authors: Ueoka‐Nakanishi Hanayo , Tsuchiya Tomohiro , Sasaki Maki , Nakanishi Yoichi , Cunningham Kyle W. , Maeshima Masayoshi
Published: October 2003

The Ca2+‐transport activity and intracellular localization of the translation product of cDNA for mung bean Ca2+/H+ antiporter (VCAX1) were examined. When the cDNA was expressed in Saccharomyces cerevisiae that lacked its own genes for vacuolar Ca2+‐ATPase and the antiporter, VCAX1 complemented the active Ca2+ transporters, and the microsomal membranes from the transformant showed high activity of the Ca2+/H+ antiporter. Treatment of the vacuolar membranes with a cross‐linking reagent resulted in a clear band of the dimer detected with antibody specific for VCAX1p. The antibody was also used for immunolocalization of the antiporter in fractions obtained by sucrose‐density‐gradient centrifugation of the microsomal fraction from mung bean. The immunostained band was detected in the vacuolar membrane fraction and the slightly heavy fractions that exhibited activity of the Golgi marker enzyme. A fusion protein of VCAX1p and green fluorescent protein was expressed in tobacco cells. The green fluorescence was clearly observed on the vacuolar membrane and, in some cases, in the small vesicles. The subcellular fractionation of transformed tobacco cells confirmed the vacuolar membrane localization of the fusion protein. These results confirm that VCAX1p functions in the vacuolar membrane as a Ca2+/H+ antiporter and also suggest that VCAX1p may exist in the Golgi apparatus.
Evidence for the involvement of the Rho GTP‐binding protein in egg activation of the ascidian Halocynthia roretzi
Authors: Toratani Satoshi , Yokosawa Hideyoshi
Published: October 2003

Eggs of the ascidian Halocynthia roretzi are activated by insemination and by treatment with calcium ionophore, leading to elevation of the vitelline coat. Here we describe the effects on egg activation of microinjection of guanosine 5′‐(γ thio) triphosphate (GTPγS, a non‐hydrolyzable GTP analog), heparin (an antagonist of the inositol 1,4,5‐trisphosphate receptor) and a monoclonal antibody to the Rho GTP‐binding protein. Microinjected GTPγS induced elevation of the vitelline coat, but not when it was co‐injected with EGTA or heparin. Pre‐injected heparin or the anti‐Rho monoclonal antibody blocked subsequent sperm‐induced elevation of the vitelline coat, but not calcium ionophore‐induced elevation. We also demonstrated that the amount of cytosolic inositol 1,4,5‐trisphosphate was increased by insemination. These results strongly suggest that the Rho GTP‐binding protein functions prior to the heparin‐blocked inositol 1,4,5‐trisphosphate receptor‐mediated Ca2+ release in the sperm induced activation process of H. roretzi eggs.
Specific expression of a gene encoding a novel calcium‐binding protein, CAF‐1, during transition of Dictyostelium cells from growth to differentiation
Authors: Abe Fumiyoshi , Maeda Yasuo
Published: October 2003

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax‐2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax‐2 cells were starved just before the putative shift (PS)‐point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin‐1 (CAF‐1) was cloned, which encoded a novel calcium‐binding protein with E‐F hand. Although CAF‐1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS‐point. Northern analysis using non‐synchronized Ax‐2 cells showed that the CAF‐1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation‐induced early expression of the CAF‐1 mRNA raised a possibility that CAF‐1 might be one of Ca2+‐binding proteins involved in the phase‐shift of cells from growth to differentiation.
Fertilization envelope formation induced by melittin in sea urchin eggs does not depend on Ca2+ signalling
Authors: Tojo Tadashi , Kamata Yasuyuki , Fujiwara Akiko , Yasumasu Ikuo
Published: October 2003

In sea urchin eggs, 10 μg/mL melittin was found to induce fertilization envelope formation without any increase in [Ca2+]i (the intracellular free Ca2+ level). On the other hand, 10 μmol/L Br‐A23187 and 100 μg/mL SDS induced fertilization envelope formation associated with [Ca2+]i increase. If EGTA was injected into eggs to make an intracellular concentration of 2 mmol/L, [Ca2+]i became quite low and was not altered by melittin, or by Br‐A23187 and SDS. In eggs containing EGTA, fertilization envelope formation was induced by melittin even in Ca2+‐free artificial sea water, but not by Br‐A23187 or SDS. Thus [Ca2+]i is essential for induction of a fertilization envelope in sea urchin eggs by Br‐A23187 or SDS but not by melittin. Melittin probably activates some Ca2+‐independent reaction downstream of Ca2+‐dependent reactions in a sequential reaction system that finally results in fertilization envelope formation.
Filamentous structure of the external surface of abalone eggs revealed by quick‐freeze deep‐etch technique
Authors: Shiroya Yoko , Sakai Yoshi T.
Published: October 2003

The external surface of abalone eggs was examined by thin section and quick‐freeze, deep‐etch electron microscopy. In thin sections, networks of fine filaments were found interconnecting the adjacent microvilli on the surface of unfertilized eggs. Quick‐freeze, deep‐etch electron microscopy revealed the three‐dimensional structure of these networks of filaments on the external surface of the egg. Mainly two networks of filaments were identified; one was composed of thicker (14–19 nm) filaments interconnecting with the neighboring microvilli nearly horizontally, and the other was composed of thinner (8–14 nm) branched filaments closely surrounding the microvilli surface as well as highly interconnecting neighboring microvilli in a polygonal pattern. The overall structure of the filamentous network on the egg surface showed no distinct alteration after fertilization. These networks of filaments observed on the egg surface may play a key role in sperm–egg interaction.
Developmental aspects of spinal cord and limb regeneration
Authors: Chernoff Ellen A. G. , Stocum David L.
Published: October 2003

The ability of birds and mammals to regenerate tissues is limited. By contrast, urodele amphibians can regenerate a variety of injured tissues such as intestine, cardiac muscle, lens and neural retina, as well as entire structures such as limbs, tail and lower jaw. This regenerative capacity is associated with the ability to form masses of mesenchyme cells (blastemas) that differentiate into the missing tissues or parts. Understanding the mechanisms that underlie blastema formation in urodeles will provide valuable tools with which to achieve the goal of stimulating regeneration in mammalian tissues that do not naturally regenerate. Here we discuss an example of tissue regeneration (spinal cord) and an example of epimorphic appendage regeneration (limb) in the axolotl Ambystoma mexicanum, emphasizing analysis of the processes that produce the regeneration blastema and of the tissue interactions and blastemal products that contribute to the regeneration‐promoting environment.
Molecular cloning of a zinc finger protein belonging to the GLI‐Krüppel family and its expression in the silk gland
Authors: Xu Pin‐Xian , Xu Xin , Suzuki Yoshiaki
Published: October 2003

We have isolated and characterized a zinc finger‐containing cDNA (ZSG‐1) from the middle silk gland of Bombyx mori. Sequence analysis indicates that the protein encoded by the ZSG‐1 transcript contains a domain of five fingers similar to the zinc finger repeats of the Xenopus transcription factor TFIIIA, the product of Drosophila segment polarity gene cubitus interruptus (ci) and the human zinc finger protein GLI, which has been found to be amplified in several human glioblastomas. The transcript expression pattern and protein distribution of ZSG‐1 during embryogenesis and silk gland development were analyzed by northern blot and immunohistochemistry. ZSG‐1 transcript was most strongly detected in the middle silk gland and weakly in the posterior silk gland at the fourth larval intermolt and molting stages. When the larvae entered the fifth intermolt, the expression of ZSG‐1 rapidly decreased by 2 days after the fourth ecdysis and maintained a low level thereafter in the middle silk gland, while the transcript in the posterior silk gland gradually became undetectable. The transcript was first detected at around stage 18 of early embryogenesis; it then increased to the maximum level by stage 27 and maintained a high level until hatching. Interestingly, the ZSG‐1 protein was most strongly distributed in the middle silk gland during embryonic silk gland development. The expression of ZSG‐1 in the silk glands suggests that this gene may play important roles in the development of silk gland.
The origin and development of retinal pigment cells and melanophores analyzed by Xenopus black‐white chimeras
Authors: Kageura Hiroshi , Eguchi Goro , Yamana Kiyotaka
Published: October 2003

At the 16 cell stage, three kinds of borealis–laevis and eight kinds of laevis–laevis chimeric embryos were produced by replacing a particular blastomere of albino embryos of Xenopus laevis with that of wild‐type embryos of X. borealis or X. laevis, and then leaving the embryos to develop into frogs.

In the borealis–laevis chimera frogs, we found that all the melanized cells (retinal pigment cells and melanophores) were derived from a transplanted wild‐type blastomere with a nuclear marker of X. borealis and that all the albino‐mutant cells derived from the host did not become melanized. Thus, retinal pigment cells and melanophores differentiated according to their own genotype. We then examined the origin of these two types of cells, using melanin as a cell‐marker in the borealis–laevis and laevis–laevis chimeras.

Retinal pigment cells derive from A1 (dorso‐animal) and A2 (latero‐animal) blastomeres. A1 of one side contributes to retinal pigment cells in both eyes. Though the blastomeres of one side contribute to the formation of bilateral melanophores, the major contribution is to melanophores of the same side. A1, A2 and V2 (latero‐vegetal) form the anterior part of the neural fold, and A2 and V2 contribute to melanophores of the head region. The most anterior part of the neural fold derived from A1 does not make a significant contribution to melanophores. Though V2 is a vegetal blastomere, it forms the anterior part of the neural fold by upward movement against the downward movement for gastrulation. A3 forms the middle and posterior parts of the neural fold and contributes to melanophores of the trunk and hindlimbs. Melanophores of hindlimbs also come from A2, A4 and V2. It is to be noted that A4 contributes to melanophores of hindlimbs, despite no apparent contribution to the neural fold.

Development of the retinal pigment cells and melanophores is discussed from the point of pigmentation patterns of the chimeras.
Monolayer culture conditions suitable for primitive erythroid cell differentiation from embryonic stem cells in vitro
Authors: Atsumi Tadao , Nagayoshi Mariko , Anzai Hiroko , Ikawa Yoji
Published: October 2003

Embryonic stem (ES) cells effectively differentiated into primitive erythroid/mesodermal cells when grown in the absence of both a feeder layer and leukemia inhibitory factor (LIF). The formation of a three‐dimensional structure, exogenous mesoderm induction factors and exogenous hematopoietic growth factors were not essential for their differentiation. Primitive erythroid cells were first detected on day 5 in the differentiation‐permissive cultures. Differentiation into other mesodermal cells was always preceded by that into primitive erythroid cells. Precursor cells of erythroid cells but of other hematoid cells were also detected in this system. This model system is useful for studying the early steps of mesoderm formation in mouse embryogenesis.
Characterization of metamorphic changes in anuran larval epidermis using lectins as probes
Authors: Amano Tosikazu , Kawabata Hiromi , Yoshizato Katsutoshi
Published: October 2003

The skin of an adult frog of Xenopus laevis was characterized by the reactivity of 20 lectins. The lectins were classified into six groups in their binding to the epidermal cells: Lycopersicon esculentum lectin (LEL)‐type which was positive for all epidermal cells; Pisum sativum agglutinin (PSA)‐type for stratum germinativum; succinylated wheat germ agglutinin (sWGA)‐type for strata spinosum, granulosum and corneum; Dolichos biflorus agglutinin (DBA)‐type for strata germinativum and spinosum; peanut agglutinin (PNA)‐type for stratum spinosum; and Ulex europaeus agglutinin (UEA‐I)‐type for strata granulosum and corneum. PSA and sWGA were utilized as markers of mitotically active germinative cells and the differentiated cells of the epidermis, respectively, to describe the metamorphic conversion of larval epidermal cells to adult type. PSA stained all epidermal cells of tadpoles before metamorphic climax. At the end of metamorphosis, PSA‐positive cells were restricted to cells in the basal layer of body epidermis while all the tail epidermis remained PSA‐positive. The other cell marker, sWGA, only stained apical cells in tadpole epidermis. During the metamorphic climax, sWGA‐positive cells appeared in the cells beneath the stratum corneum of the body region, but not in the tail region. The present study demonstrates that PSA and sWGA are useful to investigate metamorphic changes in tadpole epidermal cells.
Genes of the sea urchin embryo: An annotated list as of December 1994
Authors: Giudice Giovanni
Published: October 2003

The main literature regarding gene structure and expression in sea urchin embryos is schematically reported and briefly commented upon. Although the subject has expanded particularly over the last 10 years, to which the review mostly refers, some historical reference is also given. More space is reserved to the regulation of the synthesis of histones and cytoskeletal actins, where the attention of various authors has been especially present; the regulation of such a synthesis is described both at a territorial level and a temporal level during the sea urchin development.
Glial cell lineage in vivo in the mouse cerebellum
Authors: Miyake Toshihiko , Fujiwara Tamio , Fukunaga Takahiro , Takemura Kiyoshi , Kitamura Tadahisa
Published: October 2003

Glial cells of the cerebellum originate from cells of the ventricular germinative layer, but their lineage has not been fully elucidated. For studying the glial cell lineage in vivo by retrovirus‐mediated gene transfer, we introduced a marker retrovirus into the ventricular germinative layer of embryonic day 13 mice. In the resulting adult cerebella, virus‐labeled glial cells were grouped in discrete clusters, and statistical analysis showed that these clusters represented clones in high probability. Of 71 of the virus‐labeled glial clusters, 33 clusters were composed of astrocytes/Bergmann glia, 10 were composed of only white matter astrocytes, and 24 were composed of only oligodendrocytes. No glial clusters contained virus‐labeled neurons. These results suggest that astrocytes/Bergmann glia, white matter astrocytes and oligodendrocytes immediately arise from separate glial precursors: these three glial lineages may diverge in the course of cerebellar development.
Invasion and migration of a single chick pre‐streak stage epiblast cell in vitro: Its implication to the primitive streak formation
Authors: Toyoizumi Ryuji , Takeuchi Shigeo
Published: October 2003

To investigate the contribution of the epiblast cell behavior to the primitive streak formation, we examined the motility of a single epiblast cell from pre‐streak stage embryo in vitro. On the substratum that was evenly coated with laminin gel, epiblast cells attached well to the gel and one or a few very long and broad cellular processes protruded from their spherical cell bodies; however, they hardly locomoted on it. Unexpectedly, after overnight culture, half of the single cells dissolved the laminin gel beneath them to make well‐like holes, and invaded in the holes. On the substratum lined parallel with the fibrous laminin gels supplemented with fibronectin, they locomoted actively in accordance with the alignment. That is, they were subjected to contact guidance. In locomotion they looked like snails, extending one or a few long and broad processes in a forward direction from the spherical cell bodies. However, on the substratum lined with laminin or fibronectin only, they did not locomote actively. Individual chick pre‐streak epiblast cells had already been committed to invade, and their migratory nature existed in each cell, even though they were isolated from the epithelial sheet. The implication of these findings on the cellular basis of primitive streak formation will be discussed.
Green fluorescent protein as a marker in transgenic mice
Authors: Ikawa Masahito , Kominami Katsuya , Yoshimura Yasuhide , Tanaka Keiichi , Nishimune Yoshitake , Okabe Masaru
Published: October 2003

Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co‐factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta‐actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.
Control of trophoblast cell differentiation: Lessons from the genetics of early pregnancy loss and trophoblast neoplasia
Authors: Soares Michael J. , Chapman Belinda M. , Kamei Takayuki , Yamamoto Toshiya
Published: October 2003

Trophoblast cell differentiation is crucial to the morphogenesis of the placenta and thus the establishment of pregnancy and the growth and development of the embryo/fetus. In the present review, we discuss current evidence for the existence of regulatory genes crucial to trophoblast cell differentiation and placental morphogenesis. The elucidation of regulatory pathways controlling normal differentiation of trophoblast cells will facilitate the identification of sensitive junctures in the regulatory pathways leading to various developmental disorders, including those associated with the initiation of pregnancy, fetal growth retardation and gestational trophoblast disease.
Detection and characterization of the cis‐element in the first intron of the Ars gene in the sea urchin
Authors: Iuchi Yoshihito , Morokuma Junji , Akasaka Koji , Shimada Hiraku
Published: October 2003

Cis‐active elements that are related to temporal regulation of the Ars gene expression in embryos of the sea urchin, Hemicentrotus pulcherrimus, are localized in the region between −252 b and +38b. This promoter region, however, can only determine the onset of gene transcription at a defined period in sea urchin development, and cannot determine the level of the Ars gene transcription. The element that enhances Ars promoter activity is found in the first intron of the Ars gene. The first intron element of H. pulcherrimus shows a similar level of activity when introduced in embryos of another species of sea urchin, Temnopleurus toreumaticus. The size of the first intron element is determined to be 229 b.
The Sox gene family and its expression during embryogenesis in the teleost fish, medaka (Oryzias latipes)
Authors: Fukada Sachiko , Tanaka Minoru , Iwaya Makoto , Nakajima Masamichi , Nagahama Yoshitaka
Published: October 2003

SRY, the Y chromosomal gene, which determines testis formation during mammalian embryogenesis, encodes a potential transcription factor with a DNA binding motif known as a high mobility group I (HMG) box. We isolated HMG regions of the Sry‐related Sox gene from a medaka (Japanese killifish, Oryzias latipes) genomic library and determined their nucleotide sequences. The medaka Sox HMG box regions could be divided into four regions based on the degree of synonymous substitution and amino acid homology, and form the Sox sequence family consisting of five subgroups (Sox‐1‐1, 1‐2, 2‐1, 2‐2 and 2‐3). The deduced amino acid sequences were similar to human and mouse Sox‐1,2,3 and 14 group, one of which, SOX‐3, is located on the human X chromosome. Interestingly, we found that two marsupial Sry‐like sequences on the Y chromosome are more closely related to the medaka Sox sequences than to other mammalian Srys in the HMG box region. Genomic Southern hybridization revealed that medaka Sox sequences were present on both male and female genomes, but there was no male‐specific Sox sequence. Northern blot hybridization using medaka Sox‐1 and ‐2 PCR probes demonstrated two kinds of transcripts (2.5kb and 1.7kb). The 2.5kb transcripts were found in mature oocytes while the 1.7 kb transcripts first appeared at the gastrulation stage. The 2.5 kb and 1.7kb transcripts were both decreased 3 days after hatching.
Pre‐MPF is absent in immature oocytes of fishes and amphibians except Xenopus
Authors: Tanaka Toyomi , Yamashita Masakane
Published: October 2003

Maturation‐promoting factor (MPF), a final trigger for initiating oocyte maturation, is activated in the oocyte cytoplasm, in response to maturation‐inducing hormone (MIH) secreted from follicle cells surrounding the oocyte. MPF consists of cdc2 and cyclin B. We investigated the state of cdc2 and cyclin B in immature and mature oocytes of fishes (carp, catfish and lamprey) and amphibians (Xenopus, frog [Rana] and toad [Bufo]) using monoclonal antibodies raised against mouse cdc2, which also recognize fish and amphibian cdc2, and monoclonal antibodies against goldfish cyclin B1 and polyclonal antibodies against Xenopus cyclins B1 and B2. Anti‐cdc2 and anti‐cyclin B immunoblotting of oocyte extracts fractionated by gel filtration chromatography showed that immature oocytes from all of these species with the exception of Xenopus contained only monomeric cdc2. Cyclin B‐bound inactive cdc2 (pre‐MPF) was present only in immature Xenopus oocytes. Cdc2‐cyclin B complex was, however, found in mature oocytes from all the species examined. After the oocyte is induced to mature by MIH, cdc2 should therefore bind to cyclin B in all of these species, except Xenopus. These results suggest that the complex formation of cdc2 and cyclin B in response to MIH stimulation at the oocyte surface is a critical step for initiating oocyte maturation in fishes and amphibians, with the exception of Xenopus, in which pre‐MPF already exists in immature oocytes and only its chemical modification is required for MPF activation.
Spatial expression of the amphioxus homologue of Brachyury (T) gene during early embryogenesis of Branchiostoma belcheri
Authors: Terazawa Kouzou , Satoh Noriyuki
Published: October 2003

Amphioxus are primitive chordates that share several common anatomical features with vertebrates, such as a notochord and a dorsal nerve cord. The Brachyury (T) gene is required for notochord differentiation in vertebrates and an ascidian homologue of this gene is expressed in notochord cells at the time of developmental fate restriction. To gain insight into the molecular and developmental mechanisms underlying the evolution of chordates, we amplified a cDNA fragment for an amphioxus homologue (BbT1) of the mouse T gene, with which we examined the spatial expression of the BbT1 gene. In situ hybridization showed that BbT1 transcript was undetectable in eggs, as well as in early embryos at the cleavage and blastula stages. Distinct hybridization signals were first evident in the mesoderm of gastrulae but neither the ectoderm nor endoderm showed the signals. As development proceeded to the neurula stage, the signals became conspicuous in the primordial somite at the dorsolateral wall of the archenteron, while the primordial notochord at the dorsomedian wall of the archenteron emitted less intense hybridization signals. These results suggested that the T gene is conserved by amphioxus and that the BbT1 gene is expressed in the mesodermal region of the gastrula and neurula.
Teratogenic development in chicken embryos associated with a major deletion in the rRNA gene cluster
Authors: Delany M. E. , Taylor R. L. , Bloom S. E.
Published: October 2003

A new strain of chickens (mPNU) that segregates a severely deleted rDNA cluster was studied. Individuals heterozygous (+/p2) and homozygous (p2/p2) for the deletion were found to have 56 and 27%, respectively, of the normal complement of rRNA genes (290 copies/cell). Morphogenesis, cellular rRNA levels, and nucleolar sizes, were investigated and compared in normal +/+, +/p2, and p2/p2 embryos. Cellular rRNA contents were similar among the three genotypes at stage X, but subsequently during gastrulation, p2/p2 levels were reduced to 56% and eventually to 43% of +/+. Viability and morphogenesis were normal in p2/p2 embryos until the initial primitive streak stage of gastrulation. However, further development was abnormal and characterized by disrupted axis formation. In +/+ and +/p2 embryos, rRNA levels and nucleolar sizes increased during early development; however, the profile of these increases differed temporally and quantitatively between the genotypes. The +/p2 embryos, at the full streak stage of gastrulation, exhibited reduced rRNA levels and nucleolar sizes (80% of +/+), yet the +/p2 embryos developed normally. These studies establish a minimum copy number requirement lower than previously demonstrated, that is, a rDNA genotype with only 56% of the normal gene complement (∼160 genes) is compatible with early embryonic viability. Also, a rRNA threshold was detected: rRNA levels that were 56% of +/+ failed to support normal gastrulation; however, even under the circumstance of reduced rRNA levels (43% of control), some aspects of gastrulation apparently continue (cell migration and invagination). The teratogenic development of p2/p2 embryos is a biological consequence unique from that found in other metazoan models of rDNA‐deficiency, and will be useful as a model to investigate mechanisms of vertebrate gastrulation and axis formation.
Transforming growth factors‐α and ‐β expression in fertilized and parthenogenetic pre‐implantation mouse embryos: RNA detection with fluorescent in situ hybridization
Authors: Croteau Sylvie , Menezo Yves , Benkhalifa Moncef
Published: October 2003

Transforming growth factor (TGF)‐α and ‐β are expressed in pre‐implantation embryos and could play an important role in development. Usually, gene expression is studied using reverse transcription‐polymerase chain reaction (RT‐PCR) but here, we developed a fluorescent in situ hybridization (FISH) procedure applied on whole embryos that were spread and fixed on slides after hypotonic shock. This procedure allowed a rapid and specific detection of mRNA. For the first time, this technique was applied successfully to TGF‐α, TGF‐β1 and TGF‐β2 on parthenogenetic embryos (maternal genome only) and fertilized controls (maternal and paternal genomes). TGF‐β1 and β2 mRNA both appeared at the late 8‐cell stage and increased in morulae and blastocysts. TGF‐α expression increased from the 2‐cell stage to the blastocyst stage. Patterns of expression were similar in parthenogenetic and caryogamic embryos, suggesting that no maternal imprinting exists for these three genes.
Neuronal and mesodermal differentiation of P19 embryonal carcinoma cells is characterized by expression of specific marker genes and modulated by activin and fibroblast growth factors
Authors: van der Kruijssen Cornelia M. M. , van Achterberg Tanja A. E. , Feijen Alie , Hébert Jean M. , de Waele Peter , van den Eijnden‐van Raaij Adriana J. M.
Published: October 2003

We have used the P19 embryonal carcinoma (EC) aggregation system as a model for early mouse development to study induction and modulation of mesodermal and neuronal differentiation. By studying the expression of marker genes for differentiated cells in this model we have shown that there is a good correlation between the differentiation direction induced in P19 EC aggregates and the expression of these genes. Expression of the neuronal gene midkine is exclusively upregulated when P19 EC cells are induced to form neurons while expression of early mesodermal genes such as Brachyury T, evx‐1, goosecoid and nodal is elevated after induction to the mesodermal pathway. In the present study we have further shown that activin A blocks the different directions of differentiation of P19 EC cells induced by retinoic acid (RA) in a dose‐dependent way. To understand the mechanism behind this inhibitory action of activin A the expression of several RA‐responsive genes, including the three RA receptor genes (RARα, RARβ and RARγ) was determined. Since activin has no clear effect on the expression and activity of the RAR it is very likely that this factor acts downstream of these receptors. In addition to activin, fibroblast growth factors (FGF) were shown to modulate P19 EC cell differentiation. However, in contrast to activin, FGF exclusively blocks the mesodermal differentiation of P19 EC cells by either 10−9mol/L RA or a factor produced by visceral endoderm‐like cells (END‐2 factor). The FGF effect is dose‐independent. These results suggest an important function for RA and the END‐2 factor in the induction and for activin and FGF in the modulation of specific differentiation processes in murine development.
Ecdysteroid concentration inducing cell proliferation brings about the imaginal differentiation in the wing disc of Bombyx mori in vitro
Authors: Kawasaki Hideki
Published: October 2003

This study was undertaken to evaluate the range of 20‐hydroxyecdysone (20HE) concentrations which induce cell proliferation and imaginal differentiation in lepidopteran wing discs in vitro. Wing discs were cultured in medium containing various doses of 20HE. During imaginal differentiation in vitro, wing discs were observed histologically and the number of mitosis was counted every day. Wing discs differentiated adult features in medium containing 0.02–0.2 μg/mL 20HE, and these doses also increased the number of mitosis in disc cells. Wing discs developed the same in vitro as they do in vivo. The concentration of 20HE over 0.2 μg/mL inhibited both mitosis and imaginal differentiation. Cell proliferation, cuticle deposition and tissue elongation were successively observed in vitro the same as observed in vivo. These results suggest that a moderate concentration of ecdysteroid can induce cell proliferation followed by imaginal differentiation.
Origin of 33 kDa protein of vitelline membrane of quail egg: Immunological studies
Authors: Kuroki Masayo , Mori Makoto
Published: October 2003

The inner layer of vitelline membrane is an investment of avian ovum at the time of ovulation, but its formation is poorly understood. In order to elucidate the origin of the inner layer of vitelline membrane, a 33 kDa protein, one of the components of the inner layer, was purified from quail eggs and polyclonal antibody was raised against this protein. The tissue distribution of protein interacted with the antibody was studied by Western blotting technique. No immunoreactive component could be observed in extracts of liver, kidney, heart, lung, small intestine, brain, infundibulum, albumen‐secreting region of oviduct, uterus, and wall of small white follicles. The intensive band was detected in the granulosa layer, which was isolated from the large preovulatory follicles as a monolayer of granulosa cells sandwiched between the inner layer of vitelline membrane and the basal lamina. The granulosa cells isolated from the granulosa layer also reacted with this antibody. Theca layer had no immunoreactive components. The position of the band of the 33 kDa protein on SDS‐PAGE was sifted to higher molecular weight in follicular tissues as compared with that in the laid eggs, indicating that the structural change of the protein occurs after ovulation. These studies indicate that the material reactive to the antibody raised against a 33 kDa protein of quail vitelline membrane is synthesized by the granulosa cells.