bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential gene expression during early murine yolk sac development
Authors: Palis James , Kingsley Paul D.
Published: February 2005

The visceral yolk sac (VYS), composed of extraembryonic mesoderm and visceral endoderm, is the initial site of blood cell development and serves important nutritive and absorptive functions. In the mouse, the visceral endoderm becomes a morphologically distinct tissue at the time of implantation (E4.5), while the extraembryonic mesoderm arises during gastrulation (E6.5–8.5). To isolate genes differentially expressed in the developing yolk sac, polymerase chain reaction (PCR) methods were used to construct cDNA from late primitive streak to neural plate stage (E7.5) murine VYS mesoderm and VYS endoderm tissues. Differential screening led to the identification of six VYS mesoderm‐enriched clones: ribosomal protein L13a, the heat shock proteins hsc 70 and hsp 86, guanine‐nucleotide binding protein‐related gene, cellular nucleic acid binding protein, and ã‐enolase. One VYS endoderm‐specific cDNA was identified as apolipoprotein C2. In situ hybridization studies confirmed the differential expression of these genes in E7.5 yolk sac tissues. These results indicate that representative cDNA populations can be obtained from small numbers of cells and that PCR methodologies permit the study of gene expression during early mammalian postimplantation development. While all of the mesoderm‐enriched genes were ubiquitously expressed in the embryo proper, apolipoprotein C2 expression was confined to the visceral endoderm. These results are consistent with the hypothesis that at E7.5, the yolk sac endoderm provides differentiated liver‐like functions, while the newly developing extraembryonic mesoderm is still a largely undifferentiated tissue. © 1995 wiley‐Liss, Inc.
Transcription and localization of growth factor mRNA in the bovine oviduct
Authors: Viuff D. , Hyttel P. , Greve T. , Eriksen T. , Alexandersen S.
Published: February 2005

It has become evident that certain growth factors are involved in the regulation of the initial bovine embryogenesis. In the present study, we examined by means of Northern blot and in situ hybridization, the expression and localization in the bovine oviduct of mRNAs encoding for platelet‐derived growth factor (PDGF‐B), basic fibroblast growth factor (bFGF), and insulin‐like growth factor (IGF‐I).

Northern blot analysis on oviduct tissue demonstrated transcripts for PDGF‐B and bFGF, but not IGF‐I mRNAs. Two bands with estimated sizes of 5.0 and 1.5 kb were detected for PDGF‐B and two bands with sizes of 7.5 and 4.9 kb for bFGF. In situ hybridization analysis demonstrated localization of PDGF‐B mRNA in the lamina epithelialis and tunica muscularis of the oviduct whereas bFGF mRNA was detected in the lamina propria. It is concluded that the lamina epithelialis and lamina propria of the oviduct represent sites of synthesis of PDGF‐B and bFGF mRNA, respectively. © 1995 wiley‐Liss, Inc.
Ultrastructure of the spermatozoa and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine fish
Authors: Yao Z. , Emerson C. J. , Crim L. W.
Published: February 2005

Ultrastructure of sperm and eggs of the ocean pout (Macrozoarces americanus L.), an internally fertilizing marine teleost, was examined by scanning and transmission electron microscopy. The results showed that the sperm do not have an acrosome but have a very long mid‐piece (one to two times the sperm head length) containing numerous well‐developed elongated mitochondria. The sperm also have two tails (is biflagellate) each consisting of nine peripheral and one central pair (9 ± 2) of microtubules. This long mid‐piece and the biflagellate nature of the sperm appear to be associated with the long life‐span of the sperm and with sperm dispersal in the ovary to fertilize the eggs internally. The ocean pout eggs are enveloped by a porous chorionic membrane similar to that found in other teleosts but have two micropyles, a condition likely related to a mechanism of egg fertilization which increases the egg fertlity in the presence of low sperm numbers. Following insemination, some biochemically undefined excretions appeared on the surface of fertilized eggs and led to the acquisition of adherent capability of the eggs which formed a tightly associated egg mass in sea water. © 1995 wiley‐Liss, Inc.
Intra‐acrosomal 155,000 dalton protein increases the antigenicity during mouse sperm maturation in the epididymis: A study using a monoclonal antibody MC101
Authors: Toshimori K. , Tanii I. , Araki S.
Published: February 2005

We found an intra‐acrosomal antigen of about 155,000 daltons (155 kDa) in a survey using the monoclonal antibody MC101 raised against mouse cauda epididymal spermatozoa. Morphological studies by means of indirect immunofluorescence and immunogold electron microscopy localized the antigen to the cortex region of the anterior acrosome. Avidin biotin complex immunocytochemistry initially demonstrated a faint signal at the anterior acrosome in the testis spermatozoa that increased in intensity as the sperm moved toward the distal epididymis. This incremental immunoreactivity was also confirmed by immunoblotting following one‐dimensional SDS‐PAGE. The 155 kDa protein band was immunostained, and it was much more intense in the cauda epididymal than in the caput and corpus epididymal spermatozoa. Only a trace or no immunostain was evident in the caput or testis spermatozoa. The antigen localization did not change during passage through the epididymis, being confined at the cortex region of the anterior acrosome. The epididymal epithelial cells were not immunostained. These findings suggested that the 155 kDa protein is biochemically modified, further implying that the biochemical alteration of intra‐acrosomal material is involved in sperm maturation in the epididymis. © 1995 wiley‐Liss, Inc.
Structural and endocrine aspects of equine oocyte maturation in vivo
Authors: Grøndahl Christian , Hyttel Poul , Grøndahl Marie Louise , Eriksen Thomas , Gotfredsen Per , Greve Torben
Published: February 2005

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations.

Mares were examined every second day by transrectal ultrasonography, and follicles measuring ≤30 mm were aspirated (in vivo) using a 20‐cm‐long 12‐gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol‐17β (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON‐I) stage, 3) the peripheral flattened oocyte nucleus (PON‐II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M‐I) stage, and 6) the metaphase II (M‐II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10‐fold increase during follicle maturation, particularly associated with the progression from M‐I‐to M‐II‐stage oocytes.

The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division.

The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane‐bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well‐defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high. © 1995 wiley‐Liss, Inc.
Formation of the sea urchin male pronucleus in vitro: Membrane‐independent chromatin decondensation and nuclear envelope‐dependent nuclear swelling
Authors: Collas Philippe , Poccia Dominic L.
Published: February 2005

We demonstrate that complete sea urchin male pronuclear development in vitro is a two‐step process involving membrane‐independent chromatin decondensation and nuclear envelope‐dependent pronuclear swelling. In the absence of cytoplasmic membrane vesicles (MVs), permeabilized sperm chromatin decondenses into a spherical nucleus of ≈4 μm in diameter. Pronuclear swelling to ≈7 μm requires an intact nuclear envelope, and the degree of swelling is limited by the amount of MVs assembled on the chromatin. Furthermore, after a nuclear envelope is formed, swelling can occur in the absence of additional cytoplasmic MVs. Nuclear swelling also requires ATP hydrolysis, Ca2+ and cytosolic factors, some of which are sensitive to heat and to the sulfhy‐dryl alkylating agent, N‐ethylmaleimide. The requirement for a nuclear envelope and the rate of pronuclear swelling are consistent with previous in vivo observations. © 1995 wiley‐Liss, Inc.
Low temperature and fertilization‐induced Ca2+ changes in rat eggs
Authors: Ben‐Yosef Dalit , Oron Yoram , Shalgi Ruth
Published: February 2005

In mammalian eggs, activation by sperm that leads to resumption of meiosis is characterized by an explosive transient increase in intracellular calcium ion concentration ([Ca2+]i), followed by [Ca2+]i oscillations. In addition to the spermatozoon, various treatments can induce parthenogenetic activation, accompanied by an elevation of [Ca2+]i. It has been reported that cooling can induce egg activation, yet the mechanism of this phenomenon has not been elucidated. In the present study we followed changes in egg [Ca2+]i (measured by Fura‐2 fluorescence ratio imaging) during activation by cooling, using conditions that ensure a low rate of spontaneous activation.

Our present findings demonstrate that cooling induces egg activation as manifested by [Ca2+]i transient(s) and second polar body extrusion. Seventy‐eight of 104 eggs responded to cooling with increased [Ca2+]i. Thirty‐five percent of the responding eggs displayed a single [Ca2+]i transient, while 65% exhibited at least two [Ca2+]i transients within the time window of the experiment (30–40 min). Twenty‐two percent of these eggs displayed high‐frequency oscillations (intervals of 3.5–5.9 min). In these eggs, the overall pattern of calcium dynamics was similar to that observed in eggs activated by sperm, as judged by the transient's intervals, duration, and a gradual increase in the amplitude of successive transients. The amplitudes of [Ca2+]i transients, however, were 2–3 times lower. We propose that cooling affects [Ca2+]i homeostasis to produce fertilization‐like changes in [Ca2+]i, possibly associated with parthenogenetic activation. Moreover, great care should be exercised to prevent temperature changes during egg handling. © 1995 wiley‐Liss, Inc.
Expression of prostatic secretory protein (PSP)‐like protein in porcine corpus luteum: Isolation and characterization of a new gene encoding PSP94‐like protein
Authors: Tanaka Toshiaki , Itahana Koji , Andoh Naoki , Takeya Tatsuo , Sato Eimei
Published: February 2005

cDNA clones encoding stage‐and/or space‐specific genes in porcine ovarian follicles have been investigated by differential screening of cDNA libraries of granulosa cells and corpus luteum. A clone corresponding to mRNA preferentially expressed in the functional stage of corpus luteum was found to encode a protein similar to the human prostatic secretory protein, PSP94, which had been originally identified in male genital organs. The expression was detected widely in the functional corpora lutea by in situ hybridization histochemistry, but the signal was most prominent in the luteal cells surrounding blood vessels. In contrast, despite the structural similarity in their products, the expression of this newly identified gene was not detected in procine prostate, suggesting that these structurally related proteins play different roles in mammalian genital organs. © 1995 wiley‐Liss, Inc.
Increased accessibility of the N‐terminus of testis‐specific histone TH2B to antibodies in elongating spermatids
Authors: Unni Emmanual , Mayerhofer Artur , Zhang Yun , Bhatnagar Yogendra M. , Russell Lonnie D. , Meistrich Marvin L.
Published: February 2005

Changes in chromatin structure during spermatogenesis were investigated using a monoclonal antibody that immunoreacts with the N‐terminus of the testis‐specific histone TH2B. This monoclonal antibody, which had been raised against rat tyrosine hydroxylase (TH), cross‐reacted with TH2B because of sequence homology at the N‐termini of TH and TH2B. The epitope was localized to the N‐terminus of TH2B as trypsin‐digested chromatin which lacked the N‐terminal tail did not react with anti‐TH and preincubating anti‐TH with a synthetic peptide made from the homologous sequence between TH2B and TH inhibited its binding to TH and TH2B. In histological sections of rat testis, the primary spermatocytes and round spermatids immunoreacted weakly, whereas elongating spermatids at steps 10–12 immunoreacted intensely with anti‐TH. Increased staining of elongating spermatids was also observed in mouse and hamster by immunohistochemistry. However, immunoblotting proteins extracted from separated rat testis cells showed no increase in the TH2B content of these late steps of spermatids. The apparent increase in the immunohistochemical staining corresponds to increased accessibility of the epitope in the elongating spermatids. This indicated that the N‐terminus of TH2B is less tightly bound to DNA or to other proteins at this time in preparation for the removal of TH2B and other histones. © 1995 wiley‐Liss, Inc.
Rabbit whey acidic protein gene upstream region controls high‐level expression of bovine growth hormone in the mammary gland of transgenic mice
Authors: Thépot Dominique , Devinoy Eve , Fontaine Marie‐Louise , Stinnakre Marie‐Georges , Massoud Micheline , Kann Guy , Houdebine Louis‐Marie
Published: February 2005

Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3‐kb upstream region of the rabbit whey acidic protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley‐Liss, Inc.
Kit ligand mediates survival of type a spermatogonia and dividing spermatocytes in postnatal mouse testes
Authors: Packer A. I. , Besmer P. , Bachvarova R. F.
Published: February 2005

In the mouse testis, spontaneous death of spermatogonia has a large impact on the output of differentiating spermatids. The tyrosine kinase receptor c‐kit is expressed in type A, intermediate, and B spermatogonia, and kit‐ligand (KL) is expressed in Sertoli cells. Previous work indicated a depletion of type A spermatogonia after in vivo exposure to an antibody that blocks c‐kit function. The present work was undertaken to determine whether blocking c‐kit function results in apoptosis of spermatogonia or in an inability of spermatogonia to proliferate. Testes sections were stained by a method that detects apoptotic cells in situ. In testes of 8‐day postnatal (P8) males, type A spermatogonia are the predominant germ cell type present. Stained sections from P8 males injected with the c‐kit antagonistic antibody ACK2 showed a fivefold higher rate of cell death than uninjected controls. At least a twofold increase was observed in P12 and P30 injected males and in P30 SId + males as compared to uninjected controls. Determination of the stage of germ cell development that was affected in P30 males indicated that the frequency of gonial cell death was increased fourfold, but the frequency of death in spermatocytes around the time of the meiotic division was increased 15‐fold. It is concluded that KL acts to prevent apoptosis in the testis in vivo, that the membrane bound form of KL may be more effective, and that survival of late meiotic and dividing spermatocytes is regulated by KL through an indirect mechanism probably mediated by Sertoli cells. Thus, KL is an important regulator of spermatid output. © 1995 wiley‐Liss, Inc.
Capacitation in vitro of bovine spermatozoa by oviduct epithelial cell monolayer conditioned medium
Authors: Chian Ri‐Cheng , Lapointe Serge , Sirard Marc‐André
Published: February 2005

The effect of the active capacitating factor secreted from oviduct epithelial cell monolayers (OECM) in different environments on in vitro fertilization was evaluated. Capacitation was determined as the ability of sperm to fertilize bovine oocytes in vitro. When the mTALP was supplemented with glucose during conditioning, the sperm penetration rate was significantly reduced (P ≤ 0.05) compared to the control (7% ± 1 vs. 30% ± 4). The percentages of sperm penetrated oocytes were higher following insemination in the OECM‐conditioned medium derived from the early stage (48% ± 7) of the estrous cycle than in the OECM‐conditioned medium derived from either mid (35% ± 2) or late stages (28% ± 3) of the estrous cycle. When the medium was supplemented with 0.1 or 0.5 μg/ml estradiol‐17β during medium conditioning, sperm penetration rates increased (P ≤ 0.05) compared to the control group (55% ± 4 vs. 40% ± 3 and 54% ± 2 vs. 41% ± 3, respectively). In addition, the percentages of penetrated oocytes significantly decreased (P ≤ 0.05) following insemination when the OECM‐conditioned medium was added to 0.01%, 0.05%, and 0.1% ethanol compared to the control (25% ± 4, 19% ± 2, 18% ± 3, and 45% ± 3, respectively). Sperm penetration rates significantly (P ≤ 0.01) decreased when the OECM‐conditioned medium was heated to 100°C for 5 min (10% ± 1 vs. 40% ± 3). These results suggest that the active capacitating factor was sereted by the OECM and that this capacitating factor in the OECM‐conditioned medium was inhibited by the presence of glucose. This factor was found to be heat‐sensitive and its action was affected by ethanol. The OECM derived from the three phases of the estrous cycle as well as the presence of estradiol‐17β influenced the capacity of the OECM to secrete this capacitating factor in Vitro. © 1995 wiley‐Liss, Inc.
Allelic polymorphism in the hamster oviductin gene is due to a variable number of mucin‐like tandem repeats
Authors: Paquette Yves , Merlen Yannick , Malette Brigitte , Bleau Gilles
Published: February 2005

Oviductins are high‐molecular‐weight glycoproteins specifically secreted by the oviduct. These proteins bind to the zona pellucida of the ovulated oocyte and remain associated with the embryo during its transit in the oviduct. They may be involved in fertilization and early embryonic development. In order to explore their putative biological function, the cDNA sequence corresponding to oviductin in the golden hamster was determined. We found that the deduced amino acid sequence of this heavily O‐glycosylated protein presents characteristics typical of mucins, including serine‐ or threonine‐rich tandem repeats. Analysis of several cDNA clones and of genomic DNA revealed the presence of a single copy gene with two frequent alleles differing in the number of repeats. Comparison with oviductin sequences from other mammals indicates a high degree of conservation amongst species, except for the repeat region which shows divergence, notably in the number of repeats. Based on its biochemical and genetic properties, hamster oviductin can now be classified as a secretory mucin. This concept provides a new insight in the elucidation of its biological role: oviductin could possibly provide the oviduct and the oocyte with a protective coating ensuring normal tubal function and embryonic development. © 1995 wiley‐Liss, Inc.
Leukemia inhibitory factor, LIF receptor, and gp130 in the mouse uterus during early pregnancy
Authors: Yang Zeng‐Ming , Le Su‐Ping , Chen Dong‐Bao , Cota Juan , Siero Valentina , Yasukawa Kiyoshi , Harper Michael J. K.
Published: February 2005

Leukemia inhibitory factor (LIF) has been shown to play an important role in the implantation of mouse blastocysts. The present study was designed to investigate the changes of LIF protein, LIF receptor, and gp 130 in the mouse uterus during the early pregnancy. LIF protein and LIF receptor were at high levels in the mouse uterus near the time of ovulation and on day 4 of pregnancy. gp 130 was highest on days 3 and 4 of pregnancy. Both LIF receptor and gp 130 showed strong staining in the stroma of the day 5 uterus, at a time when LIF protein was low. The presence of LIF receptor and gp 130 in the luminal epithelium on day 4 and in the stroma on day 5 may indicate the site of the high affinity LIF receptor. The coexistence of a high level of LIF protein, LIF receptor, and gp 130 in the day 4 uterus is consistent with the previously observed high level of uterine LIF mRNA on the same day and the importance of LIF for the blastocyst implantation in mouse. © 1995 wiley‐Liss, Inc.
Effects of cumulus cells and follicle‐stimulating hormone during in vitro maturation on parthenogenetic activation of bovine oocytes
Authors: Chian R. C. , Sirard M. A.
Published: February 2005

The response to parthenogenetic activation (calcium ionophore A23187) of bovine oocytes after 30 hr of culture in different maturation conditions was evaluated. The activation rates of oocytes in response to 10 μ A23187 were similar (86% ± 4 vs. 89% ± 8) when the oocytes matured with or without cumulus cells were cultured in TCM‐199 + 10% fetal calf serum (maturation medium [MM]) alone. However, the activation rates were significantly lower in oocytes matured with than without cumulus cells (48% ± 10 vs. 79% ± 16; P < 0.05) when MM was supplemented with 0.5 μ/ml follicle‐stimulating hormone (FSH), 5.0 μg/ml luteinizing hormone (LH), and 1.0 μg/ml estradiol‐1β (E2). When oocytes with cumulus cells were cultured in different media, MM alone, MM + 0.5 μg/ml FSH, MM + 5.0 μg/ml LH, MM + 1.0 μg/ml E2, and MM + all three hormones, the percentages of activated oocytes were 91% ± 3, 52% ± 14, 83% ± 7, 83% ± 13, and 54% ± 7, respectively. Oocytes with or without cumulus cells during culture began to form a pronucleus 5 hr after activation (5% ± 1 and 7% ± 1, respectively). Finally, the proteins synthesized by oocytes matured with or without cumulus cells before (3 hr) and after (10 hr) activation was labeled with [35S]methionine for analysis. Before activation, a 47 kD protein complex was produced by cumulus cell‐intact oocytes. An active protein synthesis of 60 kD was characteristic for the oocytes with cumulus cells cultured in MM containing 0.5 μg/ml FSH. After activation, there was an appearance of proteins at 75 kD and 47 kD in the oocytes with cumulus cells but not in the oocytes without cumulus cells during the maturation period. These results indicate that bovine oocytes matured in vitro have different capacities for parthenogenetic activation depending on the presence or absence of cumulus cells and the presence of FSH in MM during culture. © 1995 wiley‐Liss, Inc.
Dynamics and organization of MAP kinase signal pathways
Authors: Errede Beverly , Cade Rebecca M. , Yashar Beverly M. , Kamada Yoshiaki , Levin David E. , Irie Kenji , Matsumoto Kunihiro
Published: February 2005

In the budding yeast, Saccharomyces cerevisiae, four separate but structurally related mitogen‐activated protein kinase (MAPK) activation pathways are known. The best understood of these regulates mating. Pheromone binding to receptor informs cells of the proximity of a mating partner and induces differentiation to a mating competent state. The MARK activation cascade mediating this signal is made up of Ste 11 (a MEK kinase [MEKK]), Ste7 (a MAPK/ERK kinase [MEK]), and the redundant MAPK‐related Fus3 and Kss1 enzymes. Another MAPK activation pathway is important for cell integrity and regulates cell wall construction. This cascade consists of Bck1 (a MEKK), the redundant Mkk1 and Mkk2 enzymes (MEKs), and Mpk1 (a MAPK). We exploited these two pathways to learn about the coordination and signal transmission fidelity of MAPK activation cascades.

Two lines of evidence suggest that the activities of the mating and cell integrity pathways are coordinated during mating differentiation. First, cells deficient in Mpk1 are susceptible to lysis when they make a mating projection in response to pheromone. Second, Mpk1 activation during pheromone induction coincides with projection formation. The mechanism underlying this coordination is still unknown to us. Our working model is that projection formation generates a mobile second messenger for activation of the cell integrity pathway.

Analysis of a STE7 mutation gave us some unanticipated but important insights into parameters important for fidelity of signal transmission. The Ste7 variant has a serine to proline substitution at position 368. Ste7‐P368 has higher basal activity than the wild‐type enzyme but still requires Ste 11 for its function. Additionally, the proline substitution enables the variant to transmit the signal from mammalian Raf expressed in yeast. This novel activity suggests that Ste7‐P368 is inherently more permissive than Ste7 in its interactions with MEKKs. Yet, Ste7‐P368 cross function in the cell integrity pathway occurs only when it is highly overproduced or when Ste5 is missing. This behavior suggests that Ste5, which has been proposed to be a tether for the kinases in the mating pathway, contributes to Ste7 specificity and fidelity of signal transmission. © 1995 wiley‐Liss, Inc.
Activation mechanism and function of the MAP kinase cascade
Authors: Gotoh Yukiko , Nishida Eisuke
Published: February 2005

MAP kinase (MAPK) and its activator, MAP kinase kinase (MAPKK), are commonly activated by a variety of extracellular stimuli in mammalian cells and in the process of Xenopus occyte maturation. In order to investigate the function of the MAPK cascade in oocyte maturation, we produced an anti‐Xenopus MAPKK which specifically reacts with MAPKK in vitro. When this antibody was microinjected into immature oocytes, MAPK activation induced by progesterone was prevented. Surprisingly, H1 kinase activation and germinal vesicle breakdown were also inhibited in the oocytes injected with this antibody. These results suggest that the MAPK cascade plays an important role in the maturation promoting factor (MPF) activation during the oocyte maturation process. When this antibody together with Mos was micoinjected into Xenopus two‐cell embryos, the Mos‐induced metaphase arrest (CSF arrest) was prevented. Thus, the MAPK cascade may mediate CSF arrest. During Xenopus early embryogenesis, a low but significant level of MAPK remains active. Injection of mRNA encoding a constitutively active MAPKK resulted in mesoderm induction in animal cap explants. In addition, fibroblast growth‐factor (FGF)‐induced mesoderm induction was inhibited by expressing CL100 (a MAP kinase phosphatase) in animal cap explants. Thus the MAPK cascade may be involved in the mesoderm induction of Xenopus embryos. The activation pathways and roles of the MAPKK/MAPK cascade in various signaling processes will be discussed. © 1995 wiley‐Liss, Inc.
Observation of optical activity in polythiourethane obtained by the controlled cationic ring‐opening polymerization of chiral cyclic thiourethane derived from serine
Authors: Nagai Atsushi , Ochiai Bungo , Endo Takeshi
Published: February 2005

The secondary structure of an optically active polythiourethane [(CH2C*H(CO2Me)NHCOS)n] was evaluated with 1H NMR, IR, and circular dichroism (CD) spectroscopy. Hydrogen bonds between the carbonyl and NH groups in the thiourethane group constrained the main chain of the chiral polythiourethane precisely under the direction of chirality in the main chain, whereas the racemic polymer was also constrained but randomly. The secondary structure of the polythiourethane in CHCl3 changed noticeably in the presence or absence of trifluoroacetic acid, which eliminated constraining intramolecular hydrogen bonds. IR spectroscopy suggested that the secondary structure was almost identical in solid and solution states. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1554–1561, 2005
Theoretical studies of growth mechanism of small fullerene cage C24 (D6d)+
Authors: Lin Wen‐Hsin , Tu Chih‐Chiang , Lee Shyi‐Long
Published: February 2005

The ring‐collapse mechanism of C24 (D6d) has been analyzed using semiempirical AM1 and B3LYP/cc‐pVDZ methods. Based on the ring‐stacking/circumscribing model, two precursors are selected. Transition states and intermediates are located and energetics are computed. Before the stacking begins, the precursor and belt reach a suitable relative orientation accompanied by the release of a large amount of energy. It is observed that the reactions between the precursors and the belts are essentially endoergic in nature, whereas the reactions between the stable intermediates and the final belts are exoergic. The deformation energies (DE) and the bond lengths R of the precursors have been computed. The DE values suggest that there is a chance of the cleavage of the bicyclic precursors as the growth process proceeds toward the cage formation. In contrast, the monocyclic precursor is found to have lower deformation energies than the bicyclic precursor. Analysis of average bond length at different cages shows that a large window is formed and the system appears to follow a cascade‐type bond formation. Comparisons are made to our previous results on C28 growth. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Mass spectrometric detection of biotinylated peptides captured by avidin functional affinity electrophoresis
Authors: Lee Bao‐Shiang , Krisnanchettiar Sangeeth , Lateef Syed Salman , Gupta Shalini
Published: February 2005

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry was used to detect biotinylated peptides captured by avidin functional affinity electrophoresis (AFAEP). Peptide samples loaded onto AFAEP were heated with sodium dodecyl sulfate to ensure that the peptides are negatively charged, and thus migrate electrophoretically toward the cathode through the embedded avidin zone in the middle of the gel. To detect the biotinylated peptides, the band containing the avidin‐biotinylated peptide complexes was excised from a 7.5% w/v native polyacrylamide gel, and biotinylated peptides were extracted with aqueous 95% v/v formamide (pH 8.2), aqueous 6 M guanidine HCl (pH 1.5), or water, at temperatures from 4 to 95°C for periods from 5 min to 24 h. It was observed that all three solvents are capable of extracting biotinylated peptides and avidin from the gel, but the best results were obtained with aqueous 95% v/v formamide (pH 8.2) at 65°C for 20 min. However, some AFAEP‐captured biotinylated peptides are not stable and are extensively modified by formamide during extraction at too high a temperature or too long an extraction time. Copyright © 2005 John Wiley & Sons, Ltd.
Multitechnique characterization of thin films of immiscible polymer systems: PS–b‐PMMA diblock copolymers and PS–PMMA symmetric blends
Authors: Kailas L. , Nysten B. , Audinot J.‐N. , Migeon H.‐N. , Bertrand P.
Published: February 2005

Immiscible polymer systems are known to form various kinds of phase‐separated structures capable of producing self‐assembled patterns at the surface. In this study, different surface characterization methods were utilized to study the surface morphology and composition produced after annealing thin polymer films. Two different SIMS techniques—static time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS) and dynamic nano‐SIMS—were used, complemented by x‐ray photoelectron spectrometry (XPS) and atomic force microscopy (AFM). Thin films (spin‐coated onto silicon wafers) of polystyrene (PS)–poly(methyl methacrylate) (PMMA) symmetric blends and diblock copolymers of similar molecular weight were investigated.

Surface enrichment by PS was found on all as‐cast samples. The samples were annealed at 160 °C for different time periods, after which the blend and the copolymer films exhibited opposite behaviour as seen by ToF‐SIMS and XPS. The annealed blend surface presented an increase in the PMMA concentration whereas that of copolymers showed a decrease in PMMA concentration compared with the as‐cast sample. For blends, the nano‐SIMS as well as AFM images revealed the formation of phase‐separated domains at the surface. The composition information obtained from ToF‐SIMS and XPS, as well as the surface mapping by nano‐SIMS and AFM, allowed us to conclude that PS formed phase separated droplet‐like domains on a thin PMMA matrix on annealing. The three‐dimensional nano‐SIMS images showed that the PS droplets were supported inside a rim of PMMA and that these droplets continued from the surface like columnar rods into the film until the substrate interface. In the case of annealed copolymer samples, the AFM images revealed topographical features resembling droplet‐like domains on the surface but there was no phase difference between the domains and the matrix. In the case of copolymers, owing to the covalent bonding between the blocks, complete phase separation was not possible. The three‐dimensional nano‐SIMS images showed domain structures in the form of striations inside the film, which were not continuous until the substrate interface. Information from the different techniques was required to gain an accurate view of the surface composition and topographical changes that have occurred under the annealing conditions. Copyright © 2005 John Wiley & Sons, Ltd.
Cortical GABAergic regulation of dopaminergic responses to psychological stress in the rat dorsolateral striatum
Authors: Matsumoto Machiko , Togashi Hiroko , Kaku Asako , Kanno Manabu , Tahara Kazue , Yoshioka Mitsuhiro
Published: February 2005

The present study was undertaken to examine the possible involvement of cortical γ‐aminobutyric acid (GABA) neuronal mechanisms in the regional differences of dopamine (DA) response to psychological stress: contextual fear conditioning (CFC) in the rat prefrontal cortex (PFC) and dorsolateral striatum (DLS). Rats that received five footshocks (shock intensity, 0.5 mA; shock duration, 2 sec) were subjected to CFC and dynamic changes in DA and GABA in both PFC and DLS were examined using dual‐probe microdialysis. Extracellular levels of DA in the PFC were enhanced during exposure to CFC, whereas the levels in the DLS were not affected by this stimulus. Extracellular levels of GABA in the PFC, but not in the DLS, were markedly enhanced by CFC. Freezing behavior observed during exposure to CFC was attenuated by the GABAA receptor antagonist bicuculline (10–3 M), which was perfused into the PFC. Intracortical application of bicuculline (10–3 M) furthermore caused sustained increases in DA levels in the DLS by CFC. These data suggest that cortical GABAA receptors contribute to modulation of DA release in the DLS in response to CFC. Thus, the GABAergic neuronal system in the PFC appears to play a key role in the regional differences of the DAergic response to psychological stress. Synapse 56:117–121, 2005. © 2005 Wiley‐Liss, Inc.
Seismic velocity structure in the Earth's outer core
Authors: Yu Wen‐che , Wen Lianxing , Niu Fenglin
Published: February 2005

We constrain seismic velocity structure in the Earth's outer core by analyzing differential travel times, waveforms, and amplitude ratios of several pairs of core phases. We use differential travel times and waveforms of PKPbc‐PKIKP at 144°–148°, PKiKP‐PKIKP at 120°–141°, PKiKP‐PKPBdiff at the PKP caustics distance range (141°–145°), and differential travel times and amplitude ratios of PKPab‐PKPbc at 146°–159°. To avoid the influence of the inner core anisotropy in velocity, we only use the PKiKP‐PKIKP and the PKPbc‐PKIKP observations whose PKIKP phases sample the inner core along the equatorial paths (paths with their ray angles being larger than 35° from the Earth's rotation axis). These observations show the following characteristics: (1) both the observed PKPbc‐PKIKP and PKiKP‐PKIKP differential travel times show a distinct “east‐west” hemispheric pattern. PKIKP phases arrive about 0.7 s earlier for those sampling the “eastern” hemisphere (40°E–180°E) than those sampling the “western” hemisphere (180°W–40°E); (2) the observed differential PKiKP‐PKPBdiff travel time residuals also exhibit a hemispheric difference. PKiKP‐PKPBdiff differential travel times are about 0.9 s larger for those sampling the western hemisphere than those sampling the eastern hemisphere; and (3) both the observed PKPab‐PKPbc differential travel times and PKPbc/PKPab amplitude ratios show scatter. Overall, these observations can be best explained by two one‐dimensional P velocity models, one for each hemisphere, at the bottom of the outer core. The seismic data sampling the eastern hemisphere can be explained by PREM at the bottom of the outer core, while those sampling the western hemisphere can be explained by a lower velocity gradient at the bottom of the outer core, which has reduced velocities relative to PREM linearly increasing from 0% at 200 km above the inner core boundary (ICB) to −0.35% at the ICB. Different velocity gradients at the bottom of the outer core indicate that there may exist a compositional difference and/or a large‐scale temperature difference there and that inner core formation processes may be different between the two hemispheres. Different inner core formation processes may produce different geometric inclusions of melt in the top of the inner core, and thus may provide an explanation to the hemispheric variation of seismic velocity and attenuation in the top of the inner core. The seismic data also suggest that the variation of seismic velocity in the tangential cylinder of the outer core, if it exists, is less than 0.1%. We use a compressional wave tomographic model to study the travel time delays caused by the seismic heterogeneities in the mantle. The tomographic model is unable to explain the scatter of the travel time data, suggesting the existence of unknown strong small‐scale seismic heterogeneities in the mantle.
Heavy ion mass loading of the geomagnetic field near the plasmapause and ULF wave implications
Authors: Fraser B. J. , Horwitz J. L. , Slavin J. A. , Dent Z. C. , Mann I. R.
Published: February 2005

The structure of the density discontinuity across the plasmapause is often based on electron and H+ density profiles with the contribution of heavy ions (He+, O+ etc) neglected. Electron and ion density measurements in this region may differ significantly due to the presence of heavy ions and it is important for the intercomparison of different datasets to understand these differences. Dynamics Explorer (DE‐1) magnetic field and plasma composition data have been used to compare heavy ion responses across the plasmapause and to calculate the mass loaded ion density (ρ) profiles. To illustrate this we investigate mass loading through radial profile variations in the Alfven velocity (VA). Results show that the gradient in ρ and VA across the plasmapause is modified when mass loading due to multiple heavy ion species is included, particularly in the presence of the O+ torus. Application to ultra‐low frequency (ULF) field line resonance is used as an example where the contribution from heavy ions smoothes out the expected ULF wave resonant frequency discontinuity at the plasmapause.
Strong electric fields from positive lightning strokes in the stratosphere
Authors: Holzworth R. H. , McCarthy M. P. , Thomas J. N. , Chin J. , Chinowsky T. M. , Taylor M. J. , Pinto O.
Published: February 2005

A balloon payload launched in Brazil has measured vector electric fields from lightning at least an order of magnitude larger than previously reported above 30 km in the stratosphere. During the flight hundreds of lightning events were recorded, including several positive cloud to ground lightning strokes. A two stroke flash, with small (15 kA peak current) and moderate (53 kA) positive strokes at a horizontal range of 34 km, produced field changes over 140 V/m at 34 km altitude. On‐board optical lightning detection, recorded with GPS timing, coupled with ground based lightning location gives high time resolution for study of the electric field transient propagation. These measurements imply that lightning electric fields in the mesosphere over large thunderstorms may be much larger than previously measured.
Statistical uncertainty associated with histograms in the Earth sciences
Authors: Vermeesch Pieter
Published: February 2005

Two types of quantitative information can be distinguished in the Earth sciences: categorical data (e.g., mineral type, fossil name) and continuous data (e.g., apparent age, strike, dip). Many branches of the Earth sciences study populations of such data by collecting a random sample and binning it into a histogram. Histograms of categorical data follow multinomial distributions. All possible outcomes of a multinomial distribution with M categories must plot on a (M − 1) simplex ΔM−1 because they are subject to a constant sum constraint. Confidence regions for such multinomial distributions can be computed using Bayesian statistics. The conjugate prior/posterior to the multinomial distribution is the Dirichlet distribution. A 100(1‐α)% confidence interval for the unknown multinomial population given an observed sample histogram is a polygon on ΔM−1 containing 100(1‐α)% of its Dirichlet posterior. The projection of this polygon onto the sides of the simplex yields M confidence intervals for the M bin counts. These confidence intervals are “simultaneous” in the sense that they form a band completely containing the 100(1‐α)% most likely multinomial populations. As opposed to categorical variables, adjacent bins of histograms containing continuous variables are not mutually independent. If this “smoothness” of the unknown population is not taken into account, the Bayesian confidence bands described above will be overly conservative. This problem can be solved by introducing an ad hoc prior of “smoothing weights” w = e−sr, where r is the integrated squared second derivative of the histogram and s is a “smoothing parameter.”
Total Ozone Mapping Spectrometer measurements of aerosol absorption from space: Comparison to SAFARI 2000 ground‐based observations
Authors: Torres O. , Bhartia P. K. , Sinyuk A. , Welton E. J. , Holben B.
Published: February 2005

The capability to detect the presence of absorbing aerosols in the atmosphere using space‐based near‐UV observations has been demonstrated in the last few years, as indicated by the widespread use by the atmospheric sciences community of the Total Ozone Mapping Spectrometer (TOMS) aerosol index as a qualitative representation of aerosol absorption. An inversion procedure has been developed to convert the unique spectral signature generated by the interaction of molecular scattering and particle absorption into a quantitative measure of aerosol absorption. In this work we evaluate the accuracy of the near‐UV method of aerosol absorption sensing by means of a comparison of TOMS retrieved aerosol single scattering albedo and extinction optical depth to ground‐based measurements of the same parameters by the Aerosol Robotic Network (AERONET) for a 2‐month period during the SAFARI 2000 campaign. The availability of collocated AERONET observations of aerosol properties, as well as Micropulse Lidar Network measurements of the aerosol vertical distribution, offered a rare opportunity for the evaluation of the uncertainty associated with the height of the absorbing aerosol layer in the TOMS aerosol retrieval algorithm. Results of the comparative analysis indicate that in the absence of explicit information on the vertical distribution of the aerosols, the standard TOMS algorithm assumption yields, in most cases, reasonable agreement of aerosol optical depth (±30%) and single scattering albedo (±0.03) with the AERONET observations. When information on the aerosol vertical distribution is available, the accuracy of the retrieved parameters improves significantly in those cases when the actual aerosol profile is markedly different from the idealized algorithmic assumption.
CB1 cannabinoid receptors are involved in neuroprotection via NF-κB inhibition
Authors: Panikashvili David , Mechoulam Raphael , Beni Sara M , Alexandrovich Alexander , Shohami Esther
Published: February 2005

We reported earlier that closed head injury (CHI) in mice causes a sharp elevation of brain 2-arachidonoylglycerol (2-AG) levels, and that exogenous 2-AG reduces brain edema, infarct volume and hippocampal death and improved clinical recovery after CHI. The beneficial effect of 2-AG was attenuated by SR141716A, a CB1 cannabinoid receptor antagonist, albeit at relatively high doses. In the present study, we further explored the role of CB1 receptors in mediating 2-AG neuroprotection. CB1 receptor knockout mice (CB1(−/−)) showed minor spontaneous recovery at 24 h after CHI, in contrast to the significant improvement in neurobehavioral function seen in wild-type (WT) mice. Moreover, administration of 2-AG did not improve neurological performance and edema formation in the CB1(−/−) mice. In addition, 2-AG abolished the three- to four-fold increase of nuclear factor κB (NF-κB) transactivation, at 24 h after CHI in the WT mice, while it had no effect on NF-κB in the CB1(−/−) mice, which was as high as in the WT vehicle-treated mice. We thus propose that 2-AG exerts its neuroprotection after CHI, at least in part, via CB1 receptor-mediated mechanisms that involve inhibition of intracellular inflammatory signaling pathways.
Role of p53 and Apoptosis in Cerebral Vasospasm after Experimental Subarachnoid Hemorrhage
Authors: Zhou Changman , Yamaguchi Mitsuo , Colohan Austin RT , Zhang John H
Published: February 2005

Our previous studies indicate that apoptosis in endothelial cells of major cerebral arteries contributes to cerebral vasospasm after subarachnoid hemorrhage (SAH). This study examined the pathologic roles of tumor suppressor p-53 in cerebral vasospasm using an established dog double-hemorrhage model. Twenty mongrel dogs were divided into four groups: (1) control, (2) SAH, (3) SAH+DMSO (vehicle), and (4) SAH+pifithrin-α (PFT) (p53 inhibitor). The p53 inhibitor (200 nmol/L) was injected into the cisterna magna daily from Day 0 through Day 3. Angiogram was performed on Day 0 and Day 7. Western blot, cell proliferation assay, histology, and TUNEL staining were conducted on the basilar arteries collected on Day 7 after SAH. The arterial diameter on Day 7 was 42%±4%, 40%±5%, and 59%±4% for SAH, SAH+DMSO, and SAH+PFT, respectively. In addition, positive staining of TUNEL and increased protein expression of p53, Bax, and PCNA in the basilar artery were observed on Day 7. PFT suppressed apoptosis in endothelial cells and proliferation in smooth muscle cells, and attenuated angiographic vasospasm. In conclusion, p53 may be a key factor in endothelial apoptosis and smooth muscle proliferation after SAH. Inhibition of p53 may potentially reduce or even prevent cerebral vasospasm.