bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Extracellular Matrices From Oocytes of the Marine Shrimp Sicyonia ingentis that Independently Mediate Only Primary or Secondary Sperm Binding
Authors: Wikramanayake Athula H. , Clark Wallis H.
Published: January 2005

During spawning, female Sicyonia ingentis simultaneously release ova and stored nonmotile sperm and mix them externally to initiate gamete interaction. Sperm bind to a thin vitelline envelope (VE) via their anterior appendage and within seconds are induced to undergo acrosomal exocytosis. The sperm penetrate the VE and become secondarily bound to the surface coat (SC), a glycocalyx on the oocyte surface. In this study, both extracellular matrices were isolated from S. ingentis oocytes. Isolated VEs mediated only primary sperm binding (i.e., before the acrosome reaction), while the isolated SCs mediated only secondary sperm binding (i.e., after acrosomal exocytosis). Isolated S. ingentis VEs were used to characterize primary sperm binding activity. The two extracellular matrices differ morphologically and possess different polypeptide profiles. Soluble fractions of isolated VEs inhibited primary sperm binding in a concentration dependent manner, and immunolocalization of VE components demonstrated highly localized VE binding sites at the tip of the sperm anterior appendage by which sperm bind eggs. Extensive Pronase digestion of VE components did not affect sperm binding activity of solubilized VE components, while complete deglycosylation with trifluoromethanesulfonic acid destroyed sperm binding activity. However, neither alkaline treatment nor enzyme digestion using glycosidases specific for asparagine and serine/ threonine linked oligosaccharides affected sperm binding activity.
ADP‐Ribosylation of Histones in Nuclei Isolated from Embryos of the Sea Urchin, Hemicentrotus pulcherrimus
Authors: Furuya Shigehisa , Kamata Yasuyuki , Yasumasu Ikuo
Published: January 2005

Two‐dimensional electrophoresis (2D‐PAGE) of a histone fraction isolated from nuclei of embryos of the sea urchin Hemicentrotus pulcherrimus exhibited almost all histone species at all stages examined. At the gastrula stage, a spot of H1A became evident and three spots closely associated with one another were found in place of a single spot of H2A.1. In the histone fraction isolated from [adenylate‐32P] NAD+‐treated nuclei of all stages examined, autoradiograms of 2D‐PAGE exhibited spots of mono [ADP‐ribosyl] ated H1 and polymodified H2B.2, H3.1, H3.3 and H4 but did not show ADP‐ribosylated H2A.1, H2A.2 or H2B.1. Poly [ADP‐ribosyl] ated H3.2, found in morulae, was not detectable in blastulae and gastrulae. Treatment with dimethylsulfate, known to activate ADP‐ribosylation in other cell types, induced poly [ADP‐ribosyl] ation of H2A.2 and H2B.1 in embryos at all stages examined, and also polymodification of H3.2 in gastrulae. ADP‐ribosylation of H1, H2B.2, H3.1 and H3.3 was hardly affected by dimethylsulfate treatment, though modification of H4 was blocked by this treatment. Probably, strong regulation of ADP‐ribosyltransferase reactions causes failures of modification of H2A.2 and H2B.1 throughout early development and also of H3.2 at the gastrula stage. Regulation of histone ADP‐ribosylation is thought to alter chromatin structures and the rate of gene expression, contributing to cell differentiation.
Independent Initiation of Calcium Dependent Glycosidase Release and Cortical Contractions during the Activation of Ascidian Eggs
Authors: Lambert Charles C. , Gonzales Genalyn P. , Miller Kimberly M.
Published: January 2005

During fertilization or ionophore induced activation, ascidian eggs rapidly release cell surface N‐acetylglucosaminidase activity used in the block against polyspermy and undergo cortical contractions before they re‐initiate meiosis. To better understand the activation process, we probed the relationship between these two processes in Ascidia ceratodes eggs by activating with different agents that increase intracellular Ca levels and under different ionic conditions. Glycosidase activity release was followed by the use of a fluorogenic substrate, and cortical contractions were followed by examining changes in cell shape with light microscopy. Ionomycin (2.7 μM) and thimerosal (1 mM) initiate glycosidase release and cortical contractions when administered in complete sea water (SW) but only the contractions in low Ca SW. Ryanodine (0.67 mM), known to raise free intracellular Ca in a number of cell types by release from the endoplasmic reticulum, causes glycosidase release but fails to initiate cortical contractions in complete SW. Thapsigargin (10 μM), which inhibits Ca dependent ATPase in the ER, causes glycosidase release but induces the contractions only about 50% of the time. These experiments show that, although glycosidase release normally precedes the ooplasmic shape changes that accompany the resumption of meiosis in ascidian eggs, they are not obligately coupled. That both processes can be induced by treatments known to raise intracellular Ca in other systems but under different conditions indicates that there may be a multiplicity of Ca requiring but functionally independent events during egg activation.
Trypsin‐like Hatching Enzyme of Mouse Blastocysts: Evidence for Its Participation in Hatching Process before Zona Shedding of Embryos6
Authors: Yamazaki Kimie , Suzuki Rika , Hojo Eriko , Kondo Shunzo , Kato Yoshihiro , Kamioka Ken , Hoshi Motonori , Sawada Hitoshi
Published: January 2005

Effects of twelve protease inhibitors on hatching of mouse embryos were investigated. Mouse hatching was strongly or moderately inhibited by trypsin inhibitors including p‐toluenesulfonyl‐Lys‐CH2Cl (TLCK) and chicken ovomucoid, while inhibitors for chymotrypsin and elastase showed weak or no inhibition. These results indicate the participation of a trypsin‐like protease in the hatching of mouse embryos as a hatching enzyme., Since TLCK is the strongest and an irreversible inhibitor for the enzyme, timing of the participation of the hatching enzyme in the hatching process was examined by pulse treatment of embryos with TLCK before and during the zona shedding. The results indicated that a trypsin‐like hatching enzyme functions before, but not during, the zona shedding of embryos, especially during a 15 h period immediately before the beginning of the shedding.
Insulin‐Induced Outgrowth of Pseudopodial Cables from Cultured Micromere‐Derived Cells Isolated from Sea Urchin Embryos at the 16 Cell Stage, with Special Reference to the Insulin‐Receptor.
Authors: Kuno Shin‐ichi , Nagura Takanori , Yasumasu Ikuo
Published: January 2005

Cultured cells derived from micromeres isolated from sea urchin embryos at the 16 cell stage are known to show outgrowth of pseudopodial cables followed by spicule rod formation when cultured in the presence of horse serum. Micromere‐derived cells cultured with bovine insulin showed pseudopodial cable growth but did not produce spicule rods. Micromere‐derived cells reversibly bound to insulin through out the period between 3 and 20 hr of culture. The dissociation constant of insulin with these cells was about 5.1 × 10−10M during the whole culture period examined. Horse serum, as well as blastocoelic fluid obtained from early gastrulae, concentration‐dependently reduced the amount of insulin bound to these cells, but the bound insulin was scarcely replaced by any proteins tested, such as bovine serum albumin. The micromere‐derived cells were bound to have an insulin‐binding protein, that may be the receptor for insulin or insulin‐like proteins. The insulin‐binding protein had a smaller molecular weight than the insulin receptor of mammalian cells. The binding of insulin with this protein in micromere‐derived cells probably results in pseudopodial cable growth.
Medaka Oocytes Rotate Within the Ovarian Follicles During Oogenesis
Authors: Iwamatsu Takashi
Published: January 2005

The purpose of the current investigation was to ascertain whether medaka oocytes rotate within the follicle. Isolated medaka follicles were incubated in modified L15 Medium for 3 hr at 26°C. During incubation, movement of oocytes within follicles held on slides under a microscope was recorded by a video cassette recorder. Within the follicle, the surface of which was marked with carbon particles, the movement of the intrafollicular oocyte was traced by dislocation of its attaching and non‐attaching filaments on the chorion. Pre‐vitellogenic oocytes exhibited rotation around the predetermined animal‐vegetal axis, accompanied by rotation at a slightly oblique angle to the axis. The velocity of oocyte rotation was about 40–48 μm hr−1 and was similar among oocytes of different stages between the pre‐vitellogenic and early vitellogenic phases of oogenesis. Rotation was inhibited by cytochalasin B treatment. Also, it was not observed in oocytes surrounded only by the granulosa cell layer when the thecal cell layer and the basement membrane were removed from the follicle. In oocytes with a thick chorion, rotation was also inhibited by impaling the oocytes with a glass needle at a right angle to the animal‐vegetal axis of the oocyte. These results provide evidence that growing medaka oocytes rotate primarily around their animal‐vegetal axis and at a slightly oblique angle to the axis. That the rotation of medaka oocytes may depend upon the movement of the granulosa and the thecal cells within the follicles was discussed.
An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.
Authors: Fausto Anna Maria , Carcupino Marcella , Mazzini Massimo , Giorgi Franco
Published: January 2005

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so‐called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.

Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane‐bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.

Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.
Identification and Characterization of Putative Receptors for Sperm‐Activating Peptide I (SAP‐I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1
Authors: Shimizu Takeshi , Yoshino Ken‐ichi , Suzuki Norio
Published: January 2005

We characterized putative receptors specific for sperm‐activating peptide I (SAP‐I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP‐I analogue [GGGY(125I)‐SAP‐I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP‐I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(125I)‐SAP‐I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)‐binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30‐residue amino‐terminal signal peptide, followed by the same sequence as the N‐terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.
The Upstream Sequence of a New Growth/Differentiation Factor, Midkine (MK), Mediates Developmentally Regulated lac Z Gene Expression in Transgenic Mice
Authors: Kaname Tadashi , Matsubara Shyuichiro , Murata Fusayoshi , Yamamura Ken‐ichi , Miyata Koichiro , Muramatsu Takashi
Published: January 2005

Midkine (MK) is the product of a retinoic acid responsive gene, and is a heparin binding protein involved in the regulation of growth and differentiation. The 1.9 kb upstream region of MK gene was fused with the bacterial β‐galactosidase gene (lac Z) and injected into fertilized mouse eggs. The resulting transgenic mice were used to evaluate the in vivo transcriptional regulation through of the upstream region. Comparison of the β‐galactosidase expression and endogenous MK expression indicated that the temporal regulation of the transgene was similar to that of MK gene expression during mouse development. The transgene was neither expressed in the preimplantation period nor in 6.5‐day embryos. Transgene expression was high and widely distributed on the 8.5th day, became restricted on the 10.5th and 12.5th days, and thereafter almost confined to the kidney. Thus, the 1.9 kb upstream region accounts for overall temporal regulation of MK gene expression, while there are some differences between the spatial regulation of the transgene expression and that of the endogenous MK gene expression. The transgene was expressed in a few limited regions of the brain of 17 day old embryos, and those sites consisted largely of matrix cells with columnar arrangements. These results suggests a role of MK in the brain development, and MK activity may be involved in retinoic acid induced malformations of the central nervous system.
Effects of K+ and the K+ Ionophore Valinomycin on the Post‐Conjugational Nuclear Differentiation of Paramecium caudatum
Authors: Mikami Kazuyuki , Ohtsu Rie
Published: January 2005

For determination of the effect of K+ on macro‐ and micronuclear differentiation Paramecium caudatum exconjugants were transferred to medium with various concentrations of Valinomycin and/or K+ at the critical stage of nuclear differentiation. The differentiation was not disturbed by transfer to medium containing 1.5 mM to 50 mM KCl. Injection of KCl solution at the critical stage also did not affect differentiation of the macronucleus appreciably. But change of the KCl concentration in the medium at the critical stage interrupted of normal development of the macronucleus.

Macro‐ and micronuclear differentiations after conjugation are known to be determined by the antero‐posterior localization of postzygotic micronuclei. This nuclear localization is achieved by elongation of mitotic spindles and marked shortening of the cell length at the time of micronuclear division. Successive measurements of cell length at 25°C showed that cells began to shorten 1.5 hr after mating‐pair separation, reaching to half the initial length about 2.5 hr after the separation, and then returning to recover their initial length within about 50 min. In a solution of K+ (50 mM) plus Valinomycin (1μg/ml or more), cell shortening was inhibited. It is not known whether elongation of mitotic spindles at the time of critical nuclear division was disordered by this treatment, but the macronuclear anlagen developed in the treated cells. Thus shortening in the cell length is not indispensable for nuclear differentiation.
Sperm Penetration of the Vitelline Envelope of Sicyonia ingentis Eggs is Mediated by a Trypsin‐like Lysin of Acrosomal Vesicle Origin
Authors: Chen Tzyy‐Ing , Green Jeffery D. , Clark Wallis H.
Published: January 2005

The sperm of the decapod crustacean Sicyonia ingentis are nonmotile, unistellate cells. At spawning, mated females release both stored sperm and eggs. The sperm bind, via the tip of their anterior appendage, to the egg's vitelline envelope (VE), rapidly undergo acrosomal exocytosis, and penetrate the VE. In the present study we used protease inhibitors to show that sperm penetration of the VE is due to the activity of a sperm trypsin‐like protease(s). Sperm extracts contained several proteases when examined using gelatin‐substrate SDS‐PAGE, with two major bands of relative molecular weight 46 kD and 30 kD. Using fluorescent peptidyl‐MCA substrates, sperm extract showed trypsin‐like and aminopeptidase‐like activities, but no chymotrypsin‐like activity. Sperm extracts were found to degrade isolated VEs. Using soybean trypsin inhibitor and anti‐inhibitor antibodies, protease was localized at the light and electron microscope levels to the acrosome remnants of reacted sperm.
A Protein That Binds an Exogastrula‐Inducing Peptide, EGIP‐D, in the Hyaline Layer of Sea Urchin Embryos
Authors: Fujita Yoshiaki , Yamasu Kyo , Suyemitsu Takashi , Ishihara Katsutoshi
Published: January 2005

Exogastrula‐inducing peptides are present in eggs and embryos of the sea urchin Anthocidaris crassispina. They induce exogastrulation when added exogenously to the embryos. In the present study, we investigated an EGIP‐D‐binding protein in the embryos. EGIP‐D was incubated with homogenates of embryos. EGIP‐D was then cross‐linked to the binding protein by use of disuccinimidyl suberate (DSS) and the complex was analyzed by western blotting with an EGIP‐D‐specific antibody. A 30‐kDa protein was detected in both eggs and embryos. To examine the localization of this protein, EGIP‐D was added to intact embryos, cross‐linked to proteins by use of DSS, and the complexes were again analyzed by western blotting. The EGIP‐D‐binding protein was detected in intact embryos but not in embryos treated with Ca2+‐ and Mg2+‐free seawater (CMF‐SW) that removes the hyaline layer (HL). It appeared, therefore, that this protein was present on the outer surface of the embryo, being a constituent of the HL. The CMF‐SW extract that contained EGIP‐D‐binding protein, inhibited the induction of exogastrulation by EGIP‐D. Furthermore, the treatment of embryos with CMF‐SW prevented EGIP‐D from inducing exogastrulation. Our observations indicate that the interaction between EGIP‐D and the binding protein is a prerequisite for induction of exogastrulation by EGIP‐D.
Retinoic Acid Treatment Induces Cell Death and the Protein Expression of Retinoic Acid Receptor β in the Mesenchymal Cells of Mouse Facial Primordia in Vitro
Authors: Motoyama Jun , Taki Keiko , Osumi‐Yamashita Noriko , Eto Kazuhiro
Published: January 2005

We isolated mesenchymal cells from individual facial primordia of mouse embryos on 11 days post coitum and examined the effects of retinoic acid (RA) on chondrogenesis, induction of cell death, and the protein expression of retinoic acid receptor (RAR) β and γ in micromass culture. Under the control condition, cells of both medial and lateral nasal prominences (MNP and LNP) displayed high chondrogenic potential, while those of maxillary and mandibular prominences (Mx and Md) had constant growth activity and low chondrogenic potential. Though none of the cells expressed detectable levels of the RAR β protein, RAR γ was expressed in the cells of all the facial primordia. One μM RA inhibited the chondrogenesis, and induced cell death accompanied with the induction of the RAR β protein in LNP, MX and Md cells within 6 hr. On the contrary, both cell death and RAR β protein induction were detected in the MNP cells treated with RA for 24 hr. These results suggest that the RAR β is involved in the process of the cell death induced by the RA treatment in the mesenchymal cells of the mouse facial primordia.
Changes in Insulin‐Binding Capacity of the Plasma Membrane Fraction during Culture in vitro of Cells Derived from Micromeres of 16‐Cell‐Stage Sea Urchin Embryos
Authors: Kuno Shin‐ichi , Mitsunaga‐Nakatsubo Keiko , Nagura Takanori , Yasumasu Ikuo
Published: January 2005

In cultured cells derived from isolated micromeres of 16‐cell stage sea urchin embryos, which undergo insulin‐induced pseudopodial cable growth, specific and reversible insulin binding by a 52‐kDa protein, probably an insulin receptor in the plasma membrane, is augmented during 5 h of culture without any change in the dissociation constant (Kuno et al : 1994). The increase in insulin‐binding capacity in micromere‐derived cells was only minimally blocked by actinomycin D and cycloheximide, which inhibited [U‐3H]uridine incorporation into RNA and [35S]methionine incorporation into protein, respectively. Insulin binding capacity was found in the plasma membrane fraction and the microsome fraction of isolated micromeres. The capacity in the plasma membrane fraction increased, accompanied by its decrease in the microsome fraction, during 5 h of culture of micromere‐derived cells. The insulin receptor is probably accumulated in microsomes of presumptive micromeres prior to the 16‐cell stage and transferred to the plasma membrane, resulting in an increase in the insulin binding capacity of micromere‐derived cells during 5 h of culture.
Vitamin A‐Mediated Homeotic Transformation of Tail to Limbs, Limb Suppression and Abnormal Tail Regeneration in the Indian Jumping Frog Polypedates maculatus
Authors: Mahapatra Pravati Kumari , Mohanty‐Hejmadi Priyambada
Published: January 2005

In this study the effects of vitamin A on tadpoles of Polypedates maculatus with an amputated tail were investigated. After amputation of half the tail at the hindlimb‐bud stage, tadpoles were exposed to vitamin A (palmitate) 10 IU/ml solution for 24 hr (Group I), 48 hr (II), 72 hr (III), 96 hr (IV), 120 hr (V) or 144 hr (VI). Vitamin A was deleterious to survival of the tadpoles since 10, 30 and 30% tadpoles died from Group IV, V and VI, respectively before the emergence of forelimbs. Regeneration of the tail was abnormal in 91.7% of the tadpoles. Vitamin A induced homeotic transformation of the amputated tail to hindlimbs in 11.6% of the animals. Normal limb development was suppressed partially or completely in 48.3% tadpoles. These results indicate that in addition to traumatic effects, vitamin A can lead to homeotic transformation of the tail into hindlimbs and can also interfere with normal development of limbs.
Developmental Potentials of Early Telencephalic Neuroepithelial Cells: A Study with Microexplant Culture
Authors: Miyata Takaki , Ogawa Masaharu
Published: January 2005

To gain a better understanding of how neuron generation begins in the telencephalon, we analyzed the developmental potentials of neuroepithelial cells (NE cells) using a microexplant culture system. By the patterns of neuron generation and cell spreading on fibronectin (FN) in vitro, we recognized at least three different types of NE cells in the early (E9 to E10.5) telencephalon of mice. Although there was no transition from one growth pattern to another in vitro, these patterns were found to represent distinct stage‐dependent profiles of the NE cells. (i) The youngest type of NE cells spread well and formed flat cell sheets on FN, but generated no neurons at all. (ii) An intermediate type showed extensive spreading on FN and generated a small number of neurons only early in culture. (iii) In contrast, NE cells at the most advanced stage examined formed compact cell sheets independent of FN and showed continuous neuron generation. These results indicate that telencephalic NE cells at the beginning of neurogenesis do not have a potential to make all cerebral neurons simultaneously. Our findings suggest that, during the period of E9 to E10.5, NE cells first acquire a potential to make the neurons produced before E12.5 and the NE cells then become capable of generating the neurons formed later under the influence of environmental factors.
Multiple Laminin Binding Proteins in Human Fetal Heart
Authors: Tomy Priya. M. , Anilkumar Narayana Panicker , Sudhakaran Perumana R.
Published: January 2005

The possibility of occurrence of laminin binding proteins in cardiac tissue under different stages of growth was examined by affinity chromatography of the soluble fraction of human fetal myocardial plasma membrane over Ln‐Sepharose. A 67 kDa protein was isolated by elution with glycine/HCl buffer containing 1 M NaCl and visualized as a coomassie stainable band on SDS gel electrophoresis under reducing conditions. Dot blot assays of the radioiodinated protein revealed the binding of 67 kDa protein with high affinity to laminin in a cation independent manner. This protein appears to be present in relatively higher amounts in tissues from early stage fetus. The occurrence of cation dependent laminin binding proteins was also examined by affinity chromatography. Electrophoresis of the EDTA eluate under reducing conditions followed by silver staining showed two prominent bands with average molecular size 130 and 174 kDa which under non‐reducing conditions appeared as two bands with average molecular weight of 115 and 135 kDa. Using radioiodinated protein in dot blot assays, its binding to Ln was found to be maximum in the presence of Mn++ ions. Immunoblotting using anti‐β1 integrin antibodies showed that 115 kDa protein is a β1 integrin suggesting the possibility of this protein belonging to the integrin group of receptors. The occurrence of multiple laminin binding proteins and the relative abundance of one of these proteins viz. the 67 kDa protein during early stages than in late stage tussue suggest a possible role for these proteins in cellular interactions with laminin during myocardial tissue development.
N‐Linked Oligosaccharides of Sea Urchin Egg Jelly Induce the Sperm Acrosome Reaction
Authors: Keller Steve H. , Vacquier Victor D.
Published: January 2005

The sea urchin egg jelly coat (EJ) induces the acrosome reaction (AR) of sperm. We previously demonstrated that a fraction of EJ containing two glycoproteins of 82‐ and 138‐kDa possess the AR inducing activity (8). Here we show that Peptide‐N‐Glycosidase‐F treatment of EJ followed by precipitation and washing in 70% ethanol results in a substantial loss of AR inducing activity in the ethanol insoluble material. When a PNGase‐F digest of EJ is chromatographed on a Sepacryl‐200 gel filtration column, an AR inducing fraction elutes within the partitioning volume. Acrosome reaction inducing activity of undigested EJ does not elute within the partitioning volume. The chromatographed AR inducing fraction of the PNGase‐F digest reacts strongly in the phenol‐sulfuric assay demonstrating carbohydrate is present; silver stained gels do not detect the presence of protein. Harsh alkaline hydrolysis of EJ in an excess of NaBH4, preserves a substantial amount of AR inducing activity. These data show that N‐linked oligosaccharides released from EJ by PNGase‐F digestion are capable of inducing the sperm acrosome reaction.
TGF‐β1 Inhibits Growth and Branching Morphogenesis In Embryonic Mouse Submandibular and Sublingual Glands in Vitro
Authors: Hardman Patricia , Landels Eleanor , Woolf Adrian S. , Spooner Brian S.
Published: January 2005

Members of the TGF‐β superfamily of polypeptides are key regulators in developmental processes. Several studies have shown that expression of TGF‐β mRNA and protein are developmentally regulated and that both are prominently expressed in tissues undergoing epithelial‐mesenchymal interactions such as branching morphogenesis. It has been shown that TGF‐β1 protein is present in E 14 mouse submandibular glands at a time when branching is already establihsed. Here we demonstrate by RT‐PCR and immunofluorescence that both TGF‐β1 mRNA and protein are present in E 13 submandibular and sublingual glands at a time when branching is being initiated. Addition of TGF‐β1 to E 13 rudiments resulted in reductions in organ size and inhibition of branching. Sensitivity to TGF‐β1 depended on the developmental stage of the rudiments (early or late E 13) and the dose of growth factor used. TGF‐β1 Also caused epithelial abnormalities, notably treated organs had elongated ducts. The effects were most pronounced in the sublingual gland. Taken together these results suggest a regulatory role for endogenous TGF‐β1 in the growth and morphogenesis of mouse salivary glands.
Calcium and Cyclic AMP Mediate Sperm Activation, but Ca2+Alone Contributes Sperm Chemotaxis in the Ascidian, Ciona savignyi
Authors: Yoshida Manabu , Inaba Kazuo , Ishida Katsumi , Morisawa Masaaki
Published: January 2005

Sperm‐activating and ‐attracting factor (SAAF) released from the ascidian, Ciona savignyi, was partially purified from egg seawater with ethanol extraction and separation with the two‐phase system of chloroform and water. SAAF did not activate sperm motility and cAMP synthesis in calcium‐free seawater (CaFSW), but activated the both in the presence of Ca2+. Sperm activation by SAAF in Ca2+‐containing medium was inhibited by flunarizine, a T‐type Ca2+channel antagonist, but L‐type Ca2+channel specific antagonists had no effect. Theophylline, a phosphodiesterase inhibitor, induced the increase of cAMP level and sperm activation in CaFSW without SAAF. On the other hand, the theophylline‐activated sperm in CaFSW did not exhibit chemotaxis toward the tip of glass capillary containing SAAF, but upon the addition of Ca2+they were attracted toward SAAF in the same manner as chemotaxis in normal artificial seawater. These results suggest that sperm activation is induced by the increased cAMP level caused by Ca2+influx through T‐type Ca2+channel, and that Ca2+alone mediates the sperm chemotaxis in Ciona.
Transient Calretinin Expression during Intervertebral Disc Formation of the Chick Embryo
Authors: Gangji Valérie , Bastianelli Enrico , Rooze Marcel , Pochet Roland
Published: January 2005

Calretinin immunoreactivity was localized during chick embryonic cervical spine development from day 4 until day 16. A transitory expression of calretinin could be seen from embryonic day 5 to embryonic day 15 in the mesenchymal cells forming the intervertebral disc. Calretinin was most abundant at embryonic day 8 when a maximal proliferation of cells occurred.

At embryonic day 12, calretinin positive fibroblasts were located along the fibers forming the annulus fibrosus. At embryonic day 16, calretinin immunoreactivity could no more be detected in the cervical column. In conclusion, an intracellular calcium binding proteins belonging to the calmodulin/troponin C superfamily, appeared to be a marker for the disc formation.
Corrected Structure of the 5′ Flanking Region of Arylsulfatase Gene of the Sea Urchin, Hemicentrotus pulcherrimus
Authors: Akasaka Koji , Sakamoto Naoaki , Yamamoto Takashi , Morokuma Junji , Fujikawa Nobuhisa , Takata Kazuko , Eguchi Sachiko , Shimada Hiraku
Published: January 2005

The nucleotide sequence of the 5′ flanking region of the arylsulfatase (Ars) gene of the sea urchin, Hemicentrotus pulcherrimus, is extensively corrected. The one previously reported from our laboratory (ref. 7, Develop, Growth & Differ., 34, 719–729, 1992) contains many sequencing errors and should be replaced with that presented here. Correction includes the addition of a 294 nucleotide (nt) sequence between −728 and −1,021. In the previous sequencing, this fragment was missed by overlooking one Hindlll site during subcloning.

In addition to a cluster of direct repeats between −2,592 and −3,440, an inverted repeat is detected between −217 and −476. A possible role of the inverted repeat in the regulation of transcription of the Ars gene is discussed.
Sensory and cognitive mechanisms for preattentive change detection in auditory cortex
Authors: Opitz B. , Schröger E. , Von Cramon D. Yes
Published: January 2005

In order to react adequately to potentially relevant information outside the focus of attention, our brain preattentively scans the acoustic environment for irregularities. Two different mechanisms are currently discussed: (i) a sensory one based on differential states of refractoriness of neurons sensitive to the features of a regular event and of neurons sensitive to features of an irregular event; (ii) a cognitive one based on a comparison of short‐lived memory representations encoding current stimulation and the invariance inherent in recent recurrent stimulation. Here, we identified regions that mediate either of the two mechanisms by combining functional magnetic resonance imaging with an experimental protocol controlling for refractoriness. The sensory mechanism was associated with activity in the primary auditory cortex, whereas the cognitive one revealed activity in nonprimary auditory areas in the anterior part of Heschl's Gyrus. Moreover, it turned out that in the traditional oddball paradigm both mechanisms contribute to irregularity detection.
Denervation‐induced alterations in gene expression in mouse skeletal muscle
Authors: Magnusson Caroline , Svensson Anna , Christerson Ulrika , Tågerud Sven
Published: January 2005

Motoneurons are important for regulating the function and properties of skeletal muscle. In the present study high‐density oligonucleotide arrays have been used to compare gene expression in innervated and six‐days denervated NMRI mouse skeletal muscle. To avoid looking at genes mainly participating in the process of atrophy, both hind‐limb muscles (atrophic after denervation) and hemidiaphragm muscle (transiently hypertrophic after denervation) were used. Only genes previously not known to respond to denervation and with potential roles in DNA/RNA interactions/transcription and/or cellular communication/signalling are presented. Data for additional genes are provided as supplementary material. Thirty‐two genes, up‐regulated by a factor of two or down‐regulated to the same extent after denervation, are presented. These include genes that may act through chromatin remodelling and/or as transcription factors/regulators (Cdkn1a, Cdr2, Hrmt1l2, Idb2, Myc/c‐myc, L‐myc1, Rb1, Sap30 and Tgif), genes possibly involved in the regulation of muscle membrane properties and/or excitation‐contraction coupling (Cacng1, Camk2d, Hrmt1l2, Kcnj12, Kcna7 and Rrad) and genes potentially involved in neuromuscular interactions and/or receptor signalling (Acvr2b, Adam19, D0H4S114, Kai1, Maged1, Mt2, Prkcabp, Ptp4a3, Ramp1, Rras, Timp1, Vegfa and Zfp145). A set of five genes with altered expression after denervation (Fzd9, Nr4a1, Frat2, Ctgf and Cyr61) indicate that Wnt signalling may be reduced in denervated skeletal muscle.
Specific pathophysiological functions of JNK isoforms in the brain
Authors: Brecht Stephan , Kirchhof Rainer , Chromik Ansgar , Willesen Mette , Nicolaus Thomas , Raivich Gennadij , Wessig Jan , Waetzig Vicki , Goetz Mario , Claussen Malte , Pearse Damien , Kuan Chia‐Yi , Vaudano Elisabetha , Behrens Axel , Wagner Erwin , Flavell Richard A. , Davis Roger J. , Herdegen Thomas
Published: January 2005

We have investigated the effect of JNK1 ko, JNK2 ko, JNK3 ko, JNK2+3 ko and c‐JunAA mutation on neuronal survival in adult transgenic mice following ischemia, 6‐hydroxydopamine induced neurotoxicity, axon transection and kainic acid induced excitotoxicity. Deletion of JNK isoforms indicated the compartment‐specific expression of JNK isoforms with 46‐kDa JNK1 as the main phosphorylated JNK isoform. Permanent occlusion of the MCA significantly enlarged the infarct area in JNK1 ko, which showed an increased expression of JNK3 in the penumbra. Survival of dopaminergic neurons in the substantia nigra compacta (SNC) following intrastriatal injection of 6‐hydroxydopamine was transiently improved in JNK3 ko and c‐JunAA mice after 7 days, but not 60 days. Following transection of the medial forebrain bundle, however, JNK3 ko conferred persisting neuroprotection of axotomised SNC neurons. None of the JNK ko and c‐JunAA mutation affected the survival of facial motoneurons following peripheral axotomy when investigated after 90 days. Finally, we determined the impact of JNK ko on the survival of animals and the degeneration of hippocampal neurons following kainic acid. JNK3 ko mice were substantially resistant against and survived kainic acid‐induced seizures. JNK3 ko and JNK1 ko showed a nonsignificant tendency for decreased or increased death of hippocampal neurons, respectively. Surprisingly, the deletion of a single JNK isoform did not attenuate the immunocytochemical signal of phosphorylated c‐Jun irrespective on the experimental set‐up. This comprehensive study provides novel insights into the context‐dependent physiological and pathological functions of JNK isoforms.
sgk1, a member of an RNA cluster associated with cell death in a model of Parkinson's disease
Authors: Stichel Christine C. , Schoenebeck Bodo , Foguet Montserrat , Siebertz Barbara , Bader Verian , Zhu Xin Ran , Lübbert Hermann
Published: January 2005

In an effort to gain deeper insight into the molecular processes underlying neurodegeneration in Parkinson's disease, we performed gene expression profiling at several early time points after MPTP‐injection into old (1‐year) mice. We used a PCR‐based gene expression profiling method, digital expression pattern display (DEPD), a method of very high sensitivity and reproducibility, which displays almost all transcripts of a tissue. To identify cell death‐associated genes, we defined clusters of differentially expressed transcripts with expression behaviour that correlated with the temporal profile of cell death progression and characterized one of these cell death clusters further. We selected one of the strongest regulated genes, the serum and glucocorticoid‐regulated kinase 1 (sgk1), and validated its differential expression by Northern blot analysis, semiquantitative PCR and in situ hybridization. Up‐regulation of sgk1 (i) coincides with the onset of dopaminergic cell death in both the 8‐week acute and 1‐year subacute MPTP models, (ii) spans the entire brain, (iii) is attenuated by the l‐deprenyl‐mediated inhibition of the MPTP conversion to its active metabolite MPP+ and (iv) is not induced by dehydration. This study demonstrated that the combination of the DEPD technology, clustering analysis and a detailed histopathology is a useful tool for elucidating molecular pathways in neurodegenerative diseases.
Dynamics of olfactory learning‐induced up‐regulation of L1 in the piriform cortex and hippocampus
Authors: Knafo Shira , Barkai Edi , Libersat Frederic , Sandi Carmen , Venero César
Published: January 2005

L1 is a cell adhesion molecule implicated in the formation of neural circuits and synaptic plasticity. We have examined the sequence and time‐frame in which modifications in the synaptic expression of L1 occur in the piriform cortex and hippocampus in the course of rule learning of an olfactory discrimination task. Rats were trained to choose the correct odour in a pair to be rewarded with drinking water. Such training requires 6–8 days on average before rats reach maximal performance. We observed a learning‐induced L1 up‐regulation that occurred at an early training stage in the piriform cortex but only after rule‐learning establishment in the hippocampus. We suggest that the dynamics of L1 up‐regulation may reflect the functional role of these brain regions in olfactory rule learning.
Static and dynamic properties of vergence‐induced reduction of ocular counter‐roll in near vision
Authors: Mandelli M.‐J. , Misslisch H. , Hess B. J. M.
Published: January 2005

We have examined the characteristics of vergence‐induced reduction of ocular counter‐roll in near vision. Monkeys were trained to make convergent and divergent refixations with the head and body either upright or in various roll orientations. During near viewing requiring 17° horizontal vergence, we found that static binocular torsion was suppressed by about 68% (averaged over both eyes, two monkeys and both near target locations). This result is in accordance with a previous study in which binocular torsion was quantified based on the displacement planes of eye positions in far and near viewing. Latency and duration of the change in torsional eye position depended (for each eye differently) on body roll and the depth plane of fixation. For instance, during convergent refixations in left‐ear‐down orientations, the latencies of the left eye were smaller and the durations were longer than those of the right eye. However, both eyes reached their final positions required to fixate the second visual target at roughly the same time. The different dynamics of the two eyes is explained by the fact that each eye rotated temporally when the eyes converged, a pattern named binocular extension of Listing's law. Coming from or aiming at a common torsional value (normal ocular counter‐roll) in convergent or divergent refixations, the required torsion differs in the two eyes. The brain compensates for these differences by adjusting the dynamics of each eye's movement.
Pharmacological antagonism of metabotropic glutamate receptor 1 regulates long‐term potentiation and spatial reference memory in the dentate gyrus of freely moving rats via N‐methyl‐d‐aspartate and metabotropic glutamate receptor‐dependent mechanisms
Authors: Naie Katja , Manahan‐Vaughan Denise
Published: January 2005

Group I metabotropic glutamate receptors (mGluRs) are critically required for multiple forms of hippocampal synaptic plasticity in vivo. The role of the receptor subtype mGluR1 in long‐term potentiation (LTP) and learning is unclear. We examined the contribution of mGluR1 to hippocampal LTP and spatial learning using the selective antagonist (S)‐(+)‐α‐amino‐4carboxy‐2‐methylbenzene‐acetic acid (LY367385). Male Wistar rats were chronically implanted with recording and stimulating electrodes to enable measurement of evoked potentials from medial perforant path–dentate gyrus granule cell synapses. An injection cannula was inserted into the ipsilateral cerebral ventricle to enable drug application. Experiments were begun 10 days after the implantation procedure. We induced a robust LTP which lasted over 25 h with a 200‐Hz tetanization. Injections of LY367385 at all concentrations under investigation (4–32 nmol in a 5‐µL injection volume) did not affect basal synaptic transmission. In contrast, we observed a dose‐dependent impairment of LTP expression: LY367385 (4 nmol) had no effect on LTP induction, whereas 8 and 16 nmol LY367385 reduced both LTP induction and expression, suggestive of an interaction with N‐methyl‐d‐aspartate receptors. We assessed the effects of daily LY367385 application (8 nmol) on performance in an eight‐arm radial maze. LY367385‐treated rats showed deficits in reference but not working memory performance compared with vehicle‐treated controls. Rearing, grooming and locomotor activity were unaffected by LY367385. These data suggest an important role for mGluR1 in LTP and learning and highlight the specific significance of this mGluR subtype for reference memory.
Inhibition of the central extended amygdala by loud noise and restraint stress
Authors: Day Heidi E. W. , Nebel Scott , Sasse Sarah , Campeau Serge
Published: January 2005

It is well established that the central nucleus of the amygdala (CEA) is involved in responses to stress, fear and anxiety. Many studies have used c‐fos expression to map the brain's response to processive stress, but curiously the CEA generally is not highly activated. We have previously shown that exposure to a novel vs. home environment reduces amphetamine‐induced activation of the lateral CEA (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BSTov). This is consistent with the idea that processive stress inhibits neurons in these nuclei. We have tested this hypothesis by exposing rats to noise, at a range of intensities from non‐stressful to stressful, or to restraint conditions, immediately after a remote injection of amphetamine, 2 mg/kg i.p., or interleukin‐1β (IL‐1β) 0.5 µg/kg i.p. (used to obtain a level of c‐fos mRNA against which to measure inhibition). In keeping with our hypothesis, amphetamine‐ or IL‐1β‐induced c‐fos and zif‐268 mRNA were significantly decreased in the CEAl and BSTov under conditions of loud noise or restraint stress compared with control conditions. This inhibition does not require a stress‐induced rise in corticosterone because data were similar in animals that had been adrenalectomized with a low‐dose corticosterone replacement. As both the CEAl and BSTov are highly γ‐aminobutyric acid (GABA) ‐ergic and project to the medial CEA (CEAm), their inhibition potentially causes an increased input to the CEAm. As the CEAm is a major output nucleus of the amygdala, this could have important consequences within the neural circuitry controlling responses to processive stress.
Reduced somatosensory hand representation in thalidomide‐induced dysmelia as revealed by fMRI
Authors: Stoeckel M. Cornelia , Jörgens Silke , Witte Otto W. , Seitz Rüdiger J.
Published: January 2005

The concept of cerebral plasticity suggests that the hand representation in somatosensory cortex is abnormal in congenital malformation disorders. To investigate this issue we studied 11 subjects with different degrees of upper extremity dysmelia due to thalidomide embryopathy in comparison to 10 control subjects. In the affected subjects fingers are typically missing in radio‐ulnar order beginning with the thumb. Haemodynamic responses to electrical stimulation of the radial‐most and ulnar‐most fingers were measured in each subject using functional magnetic resonance tomography. The size of the hand area in the primary somatosensory cortex was estimated by calculating the Euclidian distance between corresponding activation peaks on the lateral postcentral gyrus. The cortical somatosensory hand representation was found to be significantly smaller in dysmelic subjects as compared with the control subjects (P < 0.001). The shrinkage of the hand area was not proportional to the number of missing fingers. Furthermore, the cortical representation of the ulnar fingers in the dysmelic subjects was shifted towards the cortical thumb representation of the control group. We suggest that the unproportional reduction of the hand area together with the observed shift may reflect use‐dependent rather than malformation‐induced reorganization of the somatosensory hand area.
Caffeine, but not nicotine, enhances visual feature binding
Authors: Colzato Lorenza S. , Fagioli Sabrina , Erasmus Vicki , Hommel Bernhard
Published: January 2005

The distributed organization of the human visual cortex calls for a mechanism that integrates and binds the features of a perceived event, and neural synchronization is a prime candidate to serve that purpose. Animal studies suggest that synchronization in the visual cortex is enhanced by the muscarinic cholinergic system. Here we show that in healthy humans the binding of shape and colour, and of shape and location, of visual objects is increased by stimulating the muscarinic cholinergic system (caffeine consumption) but not by stimulating the nicotinic cholinergic system (nicotine consumption). Binding across perception and action is unaffected by either manipulation, suggesting a specific link between the visual system and the muscarinic cholinergic system.