bluuebunny/crossref_metadata_2025 huggingface dataset:
Rabbit blastocyst: Allocation of cells to the inner cell mass and trophectoderm
Authors: Giles J. R. , Foote R. H.
Published: February 2005

The proportion of total cells in the blastocyst allocated to the inner cell mass (ICM) and trophectoderm (TE) is important for future development and may be a sensitive indicator to evaluate culture conditions. The number of cells and their distribution within the two primary cell lineages were determined for the rabbit embryo developing in vivo after superovulation or nonsuperovulation or embryo transfer and compared with embryos developing in vitro. Comparisons were made with cultured embryos or embryos grown in vivo until 3.5, 4.0, and 4.5 days of age. Embryos from superovulated rabbits developed in vivo for 3.5, 4.0, and 4.5 days, respectively, had 361, 758, and 902 total cells (P<0.05), and in nonsuperovulated rabbits 130, 414, and 905 total cells (P<0.05), with increasing proportions of ICM cells over time (P<0.05). One‐cell embryos recovered from superovulated females and transferred to nonsuperovulated recipients developed more slowly with 70, 299, and 550 total cells after 3.5, 4.0, and 4.5 days of culture (P<0.05), respectively. The proportion of ICM cells increased with age of the embryo. Corresponding values for one‐cell embryos cultured in vitro resulted in 70, 299, and 550 total cells (P<0.05). However, in vitro culture of morula‐stage embryos in the presence of fetal bovine serum for 24 hr did not delay growth. In addition, the proportions of ICM/total cells were 0.17, 0.25, and 0.29 for embryos developing in vitro at 3.5, 4.0, and 4.5 days, respectively, similar to those for embryos developing in vivo at each of the three recovery times. These data establish for the first time the number and proportion of cells allocated to the ICM of the rabbit embryo developing in vivo or under defined conditions in vitro. © 1995 Wiley‐Liss, Inc.
Preimplantation development of mouse embryos in KSOM: Augmentation by amino acids and analysis of gene expression
Authors: Ho Yugong , Wigglesworth Karen , Eppig John J. , Schultz Richard M.
Published: February 2005

Simplex optimization has generated several media that have improved the development of mouse preimplantation embryos in vitro. One objective of this study was to compare the development of preimplantation mouse embryos in one of these computer‐optimized media, KSOM, with embryos that developed in vivo, in terms of the relative abundances of specific mRNAs involved in metabolism, transcription, and cell proliferation. First, however, since studies have indicated an improvement of other simple embryo culture media by addition of amino acids, the effects of the addition of amino acids to KSOM (KSOM/AA) on preimplantation development were assessed. We find that addition of both essential and non‐essential amino acids to KSOM augments development in vitro, as compared to development supported by KSOM without amino acids. This augmentation is observed starting at the blastocyst stage, and is associated with increased rate of development to the blastocyst stage, increased frequency of hatching, and increased number of cells in the blastocysts. Reverse‐transcription PCR was then used to assess the relative abundance of mRNAs for actin, glyceraldehyde‐3‐phosphate dehydrogenase, Na+, K+‐ATPase, Sp1, TATA box‐binding protein TBP, IGF‐I, IGF‐II, IGF‐I receptor, and IGF‐II receptor in embryos that developed in vivo and in vitro using KSOM/AA. Eight out of 9 of these mRNAs were present in the 8‐cell embryos and blastocysts raised in KSOM/AA in amounts that were indistinguishable from those in embryos that developed in vivo. It is concluded that KSOM/AA provides an environment in which preimplantation mouse embryos can undergo development that is quantitatively similar to that occurring in vivo. © 1995 Wiley‐Liss, Inc.
Transcriptional up‐regulation of the mouse gene for the muscle‐specific subunit of enolase during terminal differentiation of myogenic cells
Authors: Lamandé Noël , Brosset Sophie , Lucas Marguerite , Keller Angélica , Rouzeau Jean‐Denis , Johnson Thomas R. , Gros François , Ilan Joseph , Lazar Monique
Published: February 2005

The glycolytic enzyme enolase (EC 4.2.1.11) exists as dimers formed from three structurally related subunits α, β, and γ, encoded by separate genes. The gene encoding the β‐subunit is expressed only in striated muscles. We have previously shown that the β‐enolase gene belongs to a small subset of muscle‐specific genes showing transcriptional activity in cultured myoblasts, prior to withdrawal from the cell cycle. An increase in the level of β‐enolase mRNA occurs during terminal differentiation of myoblasts. To investigate the mechanisms underlying this increase, we have simultaneously estimated, under steady state conditions, the rate of synthesis and the stability of β‐enolase mRNA in proliferating C2.7 myoblasts as well as in differentiating myotubes. The method used is based on the isolation of newly synthesized RNA from the total RNA pool, following pulse‐labeling of intact cells in the presence of 4‐thiouridine. The results described here demonstrate a coordinate increase in newly synthesized and total β‐enolase mRNA, while the mRNA half‐life, about 4 hr, remains unchanged in the course of terminal differentiation. The expression of the gene for insulin‐like growth factor‐II (IGF‐II), a major positive regulator of myogenesis, was analyzed using the same approach.

It is concluded that the up‐regulation of β‐enolase as well as IGF‐II gene expression in differentiating muscle cells reflects an increased rate of entry of newly synthesized mRNAs into the general pool of transcripts without changes in their respective half‐lives. © 1995 Wiley‐Liss, Inc.
Rat sperm galactosyl receptor: Purification and identification by polyclonal antibodies raised against multiple antigen peptides
Authors: Mertz James R. , Banda Phillip W. , Kierszenbaum Abraham L.
Published: February 2005

We have previously reported the purification of rat testis galactosyl receptor, an equivalent to the Ca2+‐dependent (C‐type) minor variant of rat hepatic lectin‐2/3 (RHL‐2/3). We now report the purification of galactosyl receptor from rat sperm and its immunolocalization in the intact rat testis and sperm by polyclonal antibodies prepared using multiple antigen peptides (MAP) as immunogens. Two MAP antigens (designated 27‐mer and 28‐mer), corresponding to amino acid sequences of the carbohydrate‐recognition domain (galactose) and adjacent Ca2+‐binding sites of RHL‐2/3, were used for immunization. Anti‐RHL‐2/3, anti‐p27, and anti‐p28 sera crossreacted with rat hepatocyte RHL‐2/3 and its rat testis and sperm equivalent, galactosyl receptor, purified by chromatofocusing followed by galactose‐Hydropore‐EP affinity chromatography. Neither anti‐p27 nor anti‐p28 sera crossreacted with the major hepatocyte variant, RHL‐1. A RHL‐1‐equivalent was not detected in rat testis and sperm. Immunofluorescence studies demonstrated that anti‐p27 and anti‐p28 sera recognize galactosyl receptor sites at the Sertoli cell‐spermatogenic cell interface and on the dorsal surfacae of the sperm head, overlying the acrosome. The characteristic crescent‐shaped immunoreactive pattern in sperm was lost after induction of the acrosome reaction. Further studies should determine whether antisera to MAP antigens 27‐mer and 28‐mer, corresponding to specific protein motifs, can serve as immunological probes for examining cell‐cell interaction events during spermatogenesis and at fertilization. © 1995 Wiley‐Liss, Inc.
Oviductins possess chitinase‐ and mucin‐like domains: A lead in the search for the biological function of these oviduct‐specific ZP‐associating glycoproteins
Authors: Malette Brigitte , Paquette Yves , Merlen Yannick , Bleau Gilles
Published: February 2005

Over the last 10 years considerable progress has been made in the immunological and biochemical characterization of oviduct‐specific glycoproteins. It is now well established that a subclass of these secretory products, designated as oviductins, associate with the zona pellucida of the ovulated oocyte and with the early embryo. Recent reports on the cloning of cDNAs of oviductins from various species, including that of golden hamster (Mesocricetus auratus) oviductin by our laboratory, allowed us to compare their deduced amino acid sequences with those of other proteins. Optimal alignment analysis showed that oviductins contain regions of significant similarity with catalytically inactive mammalian members of the bacterial and microfilarial chitinase protein family. Most importantly, a close examination of the hamster and human deduced amino acid sequences revealed that both glycoproteins possess contiguous Ser/Thr rich repeated units, clustered in their carboxy‐terminal portions. These mucin‐type motifs are similar in the hamster and human glycoprotein, although hamster oviductin contains more of these complete units. This striking feature might indicate that these molecules play a similar role to mucin‐type glycoproteins, e.g., in protecting the oocyte and early embryo against attacks from their environment. We propose a model whereby oviductins are targeted to the oocyte via the interaction of their chitinase‐like domains with specific oligosaccharide moieties of the zona pellucida. Once localized to this structure, oviductin molecules would act as a protective shield around the oocyte and early embryo by virtue of their densely glycosylated mucin‐type domains. © 1995 Wiley‐Liss, Inc.
Expression of whey acidic protein (WAP) genes in tissues other than the mammary gland in normal and transgenic mice expressing mWAP/hGH fusion gene
Authors: Wen Jawyuan , Kawamata Yuji , Tojo Hideaki , Tanaka Satoshi , Tachi Chikashi
Published: February 2005

Whey acidic protein (WAP) is a major whey protein secreted in rodents' milk. Murine WAP (mWAP) genes have been assumed to be expressed solely in the mammary gland. However, several heterologous genes fused with the mWAP promoter and artificially introduced into animal genomes as transgene were expressed not only in the mammary gland but also in other tissues as well. In the present study, we investigated, by means of the reverse transcription polymerase chain reaction (RT‐PCR), the patterns of expression of endogenous WAP genes in tissues of normal mice and in transgenic mice carrying hGH gene coupled to the mWAP promoter sequence. The results revealed that the genes driven by the mWAP promoter, regardless of whether they are endogenous genes or transgenes, were transcribed in a variety of tissues other than the mammary gland of lactating normal female mice, although the expression levels are generally low. The expression of WAP genes in the cerebrum and the liver is regulated, as in the mammary gland, according to the reproductive stages. However, the tissue distribution of endogenous WAP gene expression in mature virgin transgenic female mice was the same as that in lactating normal female mice. © 1995 Wiley‐Liss, Inc.
Localization of nucleic acids in the nucleoli of oocytes and early embryos of mouse and hamster: An autoradiographic study
Authors: Kopečný V. , Landa V. , Pavlok A.
Published: February 2005

Mouse preovulatory oocytes, zygotes, parthenogenetically activated pronuclear oocytes, and early embryos, as well as hamster zygotes, were analyzed, by autoradiography, for the distribution of either “maternal” or newly synthesized RNAs. Early mouse embryos were also examined for the distribution of newly replicated DNA. Special attention was attributed to NLBs in oocytes or to NPBs in early embryos. In mouse oocytes, [5‐3H]uridine radioactivity accumulated (after a 2‐hr pulse) in vitro, in addition to other nuclear compartments, in the central compact material of the NLBs. There was no cytoplasmic labeling. In all parthenogenetic pronuclear embryos developed from similarly labeled oocytes, this label was distinctly detectable in the central compact material of the NPBs; less intensive labeling was seen in the nucleoplasm and cytoplasm. On the contrary, the central compact part of the mouse NPB did not show labeling in DNA after a continuous culture with [6‐3H]thymidine. In mouse and hamster pronuclear zygotes, convincing evidence was obtained for a lack of any newly synthesized nucleic acids in the compact material of NPBs using 4‐ to 10‐hr culture with [8‐3H]adenosine. Based on these data, it was shown that the NLBs of oocytes or NPBs of early embryos probably contain RNAs synthesized during the last stages of antral follicle oocyte differentiation. This unique pathway of RNAs in the oocyte—embryo system may explain the specific morphology of both oocyte and early embryo “nucleoli.” © 1995 Wiley‐Liss, Inc.
Stimulation of bovine sperm motility and respiration by the triazine dye cibacron blue F3GA
Authors: Schoff Patrick K. , First Neal L.
Published: February 2005

Bovine sperm motility and respiration were stimulated by the triazine dye Cibacron Blue F3GA (CB), which may operate as a nucleotide mimic. CB stimulation of respiration was half‐maximal at about 35 μM and respiration reached maximal levels about 1.5 minutes after CB addition. Respiratory stimulation was preceded by a transient increase in cytosolic cAMP. Sperm cAMP titers were elevated from 5 to 10 pmoles/108 cells within 30 seconds of CB addition, but rapidly dropped to a stable level of about 7.5 pmoles/108 cells. CB was a potent inhibitor of sperm membrane adenylyl cyclase and inhibited respiration in permeabilized cells. Taken together, the data indicated that CB stimulation was not manifested via the cytosol. In addition, a nonpermeant blue dextran preparation synthesized with CB also stimulated sperm respiration and motility. CB inhibited sperm membrane phosphodiesterase activity, suggesting that the transient pulse of cAMP resulted from CB interaction with this enzyme in the sperm membrane. © 1995 wiley‐Liss, Inc.
Further indications of the multicomponent nature of the acrosome reaction‐inducing substance of human follicular fluid
Authors: Fehl Petra , Miska Werner , Henkel Ralf
Published: February 2005

Human follicular fluid (hFF), which has been treated with either unspecific proteases or dextran‐coated charcoal (DCC) to remove proteins and/or steroids, cannot successfully induce the acrosome reaction (AR). After the removal of steroids, AR‐inducing activity can be restored to hFF by supplementation with exogenous progesterone, but only in the presence of intact protein. Gel filtration experiments with 3H‐progesterone‐labelled hFF showed elution of the radioactive signal in the high molecular weight range, corresponding to bound progesterone. AR‐inducing activity was seen in exactly the same fraction. Based on these results, the acrosome reaction‐inducing substance (ARIS) appears to be a complex of progesterone and a progesterone‐binding protein, which was shown to be identical with the plasma protein corticosteroid‐binding globulin (CBG) by immunological techniques. AR induction was only observed in the presence of both CBG and progesterone, suggesting a combined effect of the two components. © 1995 wiley‐Liss, Inc.
Transforming growth factor β1 expression in the endometrium of the mare during placentation
Authors: Lennard S. N. , Stewart Francesca , Allen W. R.
Published: February 2005

In situ hybridization, Northern blotting, and immunohistochemical techniques were used to study the expression of transforming growth factor beta 1 (TGFβ1) in the endometrium of the mare during the first 150 days of pregnancy (term = 330–340 days).

In situ hybridization using an oligonucleotide (45mer) probe, based on a homologous region within all known mammalian TGFβ1 DNA sequences, demonstrated TGFβ1 mRNA accumulation in the glandular and lumenal epithelial cells of the endometrium from day 33 onwards which corresponds to the time of implantation (day 33–45). Expression in the endometrium remained at a high level to the end of the sampling period (day 150). There was also marked expression of TGFβ1 in the mononuclear cells accumulated around the periphery of the specialized trophoblast cells of the endometrial cup within the endometrium and in the mononuclear cells accumulated in the endometrial stroma of mares carrying failing donkey‐in‐horse pregnancies created by embryo transfer. The sense (control) oligonucleotide probe exhibited no hybridization to any tissue at any stage. Northern blot analysis demonstrated that the oligonucleotide probe and a porcine TGFβ1 cDNA clone hybridized to a single 2.5 kb transcript in horse endometrial and lymphocyte RNA, thus validating the oligonucleotide probe for detection of horse TGFβ1 mRNA. Furthermore, both probes demonstrated an increased signal in the pregnant endometrium from day 33 onwards, thereby confirming the in situ hybridization results. Immunostaining with a specific anti‐bovine TGFβ1 serum also showed increasing TGFβ1 accumulation in endometrial epithelia during pregnancy, and localization of the protein in endometrial stroma and in the trophoblast layer of the placenta after day 60 of pregnancy.

These results show that TGFβ1 expression increases in the maternal endometrium of the mare at the time of implantation and that it may play a role in regulating endometrial and/or trophoblast growth and differentiation during placentation in this species. It may also influence fetal development (via placental transfer) at a later stage of gestation. © 1995 wiley‐Liss, Inc.
Mouse androgen‐dependent epididymal glycoprotein crisp‐1 (DE/AEG): Isolation, biochemical characterization, and expression in recombinant form
Authors: Eberspaecher Uwe , Roosterman Dirk , Krätzschmar Jörn , Haendler Bernard , Habenicht Ursula‐Friederike , Becker Andreas , Quensel Christina , Petri Thomas , Schleuning Wolf‐Dieter , Donner Peter
Published: February 2005

In the rat, the secretory glycoprotein DE/AEG is one of the main constitutents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine‐rich secretory protein‐1 (CRISP‐1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192–198). The protein has now been isolated from the same organ and its N‐terminal amino acid sequence has been determined. CRISP‐1 exhibited an iso‐electric point of ∼6.8. High levels of CRISP‐1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP‐1 represented ∼15% of the total protein. For heterologous expression, the CRISP‐1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co‐transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP‐1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N‐terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein. © 1995 wiley‐Liss, Inc.
Mouse embryonic stem cells express receptors of the insulin family of growth factors
Authors: Shi C. Z. , Dhir R. N. , Kesavan P. , Zhang S. L. , Matschinsky F. M. , Heyner S.
Published: February 2005

Insulin and insulin‐like growth factors (IGF‐I and ‐II) are members of a family of growth factors which are known to be developmentally regulated during preimplantation mouse embryogenesis. The physiological actions of the insulin family of growth factors are mediated by interactions with specific cell surface receptors that are detectable on the cells of preimplantation mouse embryos. Mouse embryonic stem (ES) cells are totipotent cells derived directly from the inner cell mass of the blastocyst. ES cells have the ability to differentiate into all three germ layers and have unlimited growth potential under certain culture conditions. The great advantage of ES cells is the ability to obtain large amounts of tissue for biochemical studies as compared with preimplantation embryos. To examine in greater detail the biological actions of the insulin family of growth factors, the expression of their cognate receptors on ES cells was examined. ES cells were cultured in DMEM medium supplemented with leukemia inhibitory factor (LIF) to maintain the undifferentiated state. Receptor expression was evaluated at the mRNA level using the reverse transcription polymerase chain reaction (RT‐PCR), and at the protein level by radioactive labeled ligand‐receptor binding assay. Using RT‐PCR, mRNAs of all three growth factor receptors were detected in ES cells. Messenger RNA from ES cells was reverse transcribed into cDNA by AMV reverse transcriptase at 42°C for 1 hr. The reverse transcription reaction was amplified with Taq polymerase and specific primers for insulin, IGF‐I, or IGF‐II receptors by PCR. RT‐PCR and the control plasmid cDNA PCR products were resolved electrophoretically on 3% agarose gels. Each amplified PCR product showed the predicted correct size. The target sequence of RT‐PCR amplified fragments were further verified by restriction enzyme digestion. The expression of receptors at the protein level was confirmed by Scatchard analysis, which showed specific binding of the radiolabeled ligands. This study shows that ES cells may provide a useful model to study the biological actions of the insulin family growth factors. © 1995 wiley‐Liss, Inc.
Xenopus lipovitellin 1 is a Zn2+‐ and Cd2+‐binding protein
Authors: Sunderman F. William , Antonijczuk Katarzyna , Antonijczuk Adam , Grbac‐Ivankovic Svetlana , Varghese Alison H. , Korza George , Ozols Juris
Published: February 2005

This report discusses the identification of a Zn2+‐ and Cd2+‐binding protein of Xenopus laevis that is abundant in vitellogenic oocytes and in embryos from fertilization to stage 46. Oocyte or embryo homogenates were fractionated by SDS‐PAGE, blotted onto nitrocellulose, and probed with 65Zn2+ or 109Cd2+. The resulting autoradiograms showed binding of both radio‐nuclides to a protein, designated pCdZn. Freon extraction of oocyte and embryo homogenates showed pCdZn to be a yolk protein. When pCdZn was isolated from oocyte homogenates by ammonium sulfate precipitation, delipidation, and chromatography, it co‐purified with lipovitellin 1. The amino acid composition of pCdZn closely resembled the reported composition of lipovitellin 1 and the molecular weight of purified pCdZn (∼115 kD) corresponded to reported values for lipovitellin 1 (111–121 kD). Amino acid sequence analyses of five peptides derived from pCdZn yielded 94% identity to the reported sequence of lipovitellin 1, deduced from the DNA sequence of the Xenopus vitellogenin A2 precursor gene. Based on these findings, pCdZn was identified as lipovitellin 1. This study suggests that lipovitellin 1 is the major storage protein for zinc in mature oocytes and developing embryos of Xenopus laevis. © 1995 wiley‐Liss, Inc.
Carbendazim (MBC) disrupts oocyte spindle function and induces aneuploidy in hamsters exposed during fertilization (meiosis II)
Authors: Zuelke Kurt A. , Perreault Sally D.
Published: February 2005

Peri‐fertilization exposure to Carbendazim (MBC; a microtubule poison) induces infertility and early pregnancy loss in hamsters. Presently, both in vivo and in vitro techniques were employed to characterize the effects of MBC on cellular aspects of fertilization in hamsters. Exposure to MBC during either in vivo or in vitro fertilization (IVF) induced identical morphological abnormalities in the maternal chromatin of zygotes and embryos. These abnormalities included either multiple second polar bodies (PB2), and/or multiple small female pronuclei (PN), or meiotic arrest. Multiple PB2, multiple female PN, multiple PB2 with multiple female PN, or meiotic arrest were exhibited by approximately 31%, 15%, 12%, and 2% of the in vivo zygotes; and 3%, 16%, 36%, and 20% of IVF zygotes, respectively. The effects of MBC persisted to day 2 of pregnancy as indicated by decreased (P ≤ 0.05) embryo development to the two‐cell stage and the presence of micronuclei in 6% of two‐cell embryos from MBC‐treated females. Immunofluorescence analysis of microtubules (MTs) confirmed that MBC disrupted spindle MTs during IVF. Numerical chromosome analysis revealed that a single dose of MBC administered during in vivo fertilization induced aneuploidy in the resulting pronuclear‐stage zygotes. The present data point to two mechanisms by which peri‐fertilization MBC exposure may induce early pregnancy loss: 1) arrested meiosis with no zygotic cleavage; or 2) induction of zygotic aneuploidy with subsequent developmental arrest. © 1995 wiley‐Liss, Inc.
Analysis of the motility parameters of in vitro hyperactivated hamster spermatozoa
Authors: Shivaji S. , Peedicayil J. , Devi L. Girija
Published: February 2005

Golden hamster cauda epididymal spermatozoa under in vitro capacitating conditions exhibited time‐dependent transformation of motility pattern, and the frequency of occurrence of a particular motility type was found to be dependent on the depth of the motility chamber used. The nonhyperactivated spermatozoa with planar motility were the most predominant at 0 hr irrespective of the depth of the motility chamber. But spermatozoa with the helical motility pattern were not detectable up to 6 hr when the Makler chamber was used, whereas in both the slide chamber and cannula, by 2 hr such spermatozoa constituted 90% of the total spermatozoa. However, by 6 hr the hyperactivated circular moving spermatozoa were the predominant type in all the chambers. Sperm motility chamber depths were also found to effect the motility parameter values of hamster spermatozoa, but this effect was also found to be dependent on the type of motility. Increase in chamber depth did not alter any of the motility parameter values of spermatozoa with hatchet type of motility and only increased the amplitude of lateral head displacement (ALH) in planar type. But in spermatozoa with the helical type of motility, an increase in chamber depth increased the progressive velocity (VSL), path velocity (VAP), curvilinear velocity (VCL), straightness (STR), linearity (LIN), and ALH. In spermatozoa with the circular type of motility, an increase in VSL, VAP, VCL, and ALH was also observed, but STR and LIN decreased. The hyperactivated spermatozoa could be distinguished from the nonhyperactivated spermatozoa because the former were swimming in circles, had low progressive velocity, decreased straightness and linearity of path, and also exhibited an increase in the amplitude of lateral head displacement compared to the nonhyperactivated spermatozoa. Further, the spermatozoa with helical motility could be differentiated from the nonhyperactivated, circular, and hatchet spermatozoa in that they had the highest VSL, VAP, VCL, and ALH. Spermatozoa with hatchet movement were slow and exhibited very low STR and LIN. Thus the motility parameters could be used to distinguish the nonhyperactivated and hyperactivated distal cauda epididymal spermatozoa. © 1995 wiley‐Liss, Inc.
Bovine cumulus cell expansion does not depend on the presence of an oocyte secreted factor
Authors: Ralph J. H. , Telfer E. E. , Wilmut I.
Published: February 2005

Communication between the oocyte and its somatic cells has been shown to be important in oocyte development. Here we examined how the oocyte may be involved in bovine cumulus cell expansion. Intact bovine cumulus oocyte complexes (COC) were obtained by puncturing antral follicles. From the intact COC, oocytectomised complexes (OOX) were produced by micro surgical removal of the oocyte. Clumps of cumulus cells (CC) were obtained by micro‐dissection. Intact or OOX complexes or CC were matured in the presence of fetal calf serum and hFSH (6 mlU/ml) for 24 hr and the degree of expansion measured. The presence of the oocyte is not essential to allow bovine cumulus expansion to occur as expansion occurred in all groups. Murine OOX complexes from eCG primed 35–40‐day‐old C57BL6/CBA F1 hybrids (known to require the presence of an oocyte secreted factor for cumulus expansion) were cultured with or without denuded bovine oocytes (1 oocyte/μl). Murine OOX complexes expanded only in the presence of denuded bovine oocytes. Thus some factor produced by bovine oocytes enabled expansion of murine OOX complexes. To determine whether the factor is secreted by bovine oocytes, murine OOX were cultured with or without media conditioned by bovine oocytes (1 oocyte/μl for 4 hr). Significant expansion of murine OOX occurred in media conditioned by bovine oocytes. This shows that the cumulus expansion enabling effect of bovine oocytes is released into the surrounding media. Media conditioned by bovine oocytes and then frozen for up to 1 month showed that the activity by the factor can withstand freezing. © 1995 wiley‐Liss, Inc.
Intracellular calcium reaches different levels of elevation in hyperactivated and acrosome‐reacted hamster sperm
Authors: Suarez Susan S. , Dai Xiaobing
Published: February 2005

Calcium plays a role in sperm motility hyperactivation and the acrosome reaction, but the relationship between cytoplasmic calcium (Ca2+ in) levels in the two states was heretofore unknown. The Ca2+ indicator indo‐1 was used to detect Ca2+in in moving hamster sperm in two sets of experiments. In the first experiment, activated, hyperactivated, and zona pellucida‐induced acrosome‐reacted/hyperactivated sperm were analyzed at the time of peak of activity for each state. In the second experiment, sperm in all states were analyzed at one time point. In both sets, mean Ca2+in in the acrosomal region, postacrosomal region, and flagellar midpiece was greater in hyperactivated sperm than in activated sperm, and in acrosome‐reacted/hyperactivated sperm than in unreacted/hyperactivated sperm (P ≤ 0.001). Ca2+in had increased to a greater extent in the midpiece than in the head in hyperactivated sperm, while the reverse was true for acrosome‐reacted sperm. Oscillations at the frequency of the flagellar beat cycle were detected chiefly in the proximal flagellar midpiece of acrosome‐reacted sperm, as they had been previously reported to occur in activated and hyperactivated sperm. Thus, Ca2+in may be maintained at two different elevated levels in sperm, and continues to oscillate after the acrosome reaction. © 1995 wiley‐Liss, Inc.
Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids of human spermatozoa
Authors: Alvarez Juan G. , Storey Bayard T.
Published: February 2005

Intact human sperm incorporated radiolabelled fatty acids into membrane phospholipids when incubated in medium containing bovine serum albumin as a fatty acid carrier. The polyunsturated fatty acids were preferentially incorporated into the plasmalogen fraction of phospholipid. Uptake was linear with time over 2 hr; at this time sufficient label was available to determine the loss of fatty acids under conditions of spontaneous lipid peroxidation. Loss of the various phospholipid types, the loss of the various fatty acids from these phospholipids, and the overall loss of fatty acids were all first order. The loss of saturated fatty acids was slow with first order rate constant k1 = 0.003 hr−1; for the polyunsaturated fatty acids, arachidonic and docosahexaenoic acids, k1 = 0.145 and 0.162 hr−1, respectively. The rate of loss of fatty acids from the various phospholipid types was dependent on the type, with loss from phosphatidylethanolamine being the most rapid. Among the phospholipid types, phosphatidylethanolamine was lost at the greatest rate. Analysis of fatty acid loss through oxidation products was determined for radiolabelled arachidonic acid. Under conditions of spontaneous lipid peroxidation at 37°C under air in the absence of albumin, free arachidonic acid was found in the medium, along with minor amounts of hydroxylated derivative. All the hydroperoxy fatty acid remained in the cells. In the presence of albumin, all the hydroperoxy fatty acid was found in the supernatant bound to albumin; none could be detected in the cells. Albumin is known as a very potent inhibitor of lipid peroxidation in sperm; its action may be explained, based on these results, as binding the damaging hydroperoxy fatty acids. These results also indicate that a phospholipase A2 may act in peroxidative defense by excising a hydroperoxy acyl group from phospholipid and providing the hydroperoxy fatty acid product as substrate to glutathione peroxidase. This formulation targets hydroperoxy fatty acid as a key intermediate in peroxidative degradation. © 1995 wiley‐Liss, Inc.
Identification of a circulatory and oocytic avian apolipoprotein D
Authors: Vieira Amandio V. , Lindstedt Ken , Schneider Wolfgang J. , Vieira Päivi M.
Published: February 2005

Apolipoprotein D (apo D) is an unusual apolipoprotein with respect to structure and sites of synthesis. It has been identified in the circulatory system of certain mammals, but its physiological role remains poorly understood. In this report, it is shown that apo D is not exclusively a mammalian apolipoprotein, and evidence is presented which suggests a novel function for this protein during oogenesis in the chicken. The avian apo D which we identify has the same molecular mass (29 kDa) as the human protein and also associates preferentially with the plasma lipoprotein fraction. In addition to the 29 kDa avian apo D species, an immunoreactive 24 kDa protein is observed in chicken serum. The chicken apo D (along with the 24 kDa species) is also demonstrated to be present in the yolk of the rapidly growing chicken oocyte, a cell with high endocytic activity. Clathrin‐coated vesicles from chicken oocytes, which we have previously shown to contain specific lipoproteins along with their oocytic receptors (Bujo et al., 1994: EMBO J 13:5165–5175), also contain chicken apo D. Thus, apo D represents a novel candidate for plasma‐to‐oocyte transport of lipids and/or their mobilization during embryogenesis in oviparous species. © 1995 wiley‐Liss, Inc.
Transcriptional regulation by MAP kinases
Authors: Davis Roger J.
Published: February 2005

Tyrosine kinase growth factor receptors activate MAP kinase by a complex mechanism involving the SH2/3 protein Grb2, the exchange protein Sos, and Ras. The GTP‐bound Ras protein binds to the Raf kinase and initiates a protein kinase cascade that leads to MAP kinase activation. Three MAP kinase kinase kinases have been described‐c‐Raf, c‐Mos, and Mekk—that phosphorylate and activate Mek, the MAP kinase kinase. Activated Mek phosphorylates and activates MAP kinase. Subsequently, the activated MAP kinase translocates into the nucleus where many of the physiological targets of the MAP kinase signal transduction pathway are located. These substrates include transcription factors that are regulated by MAP kinase phosphorylation (e.g., Elk‐1, c‐Myc, c‐Jun, c‐Fos, and C/EBPβ). Thus the MAP kinase pathway represents a significant mechanism of signal transduction by growth factor receptors from the cell surface to the nucleus that results in the regulation of gene expression.

Three MAP kinase homologs have been identified in the rat: Erk1, Erk2, and Erk3. Human MAP kinases that are similar to the rat Erk kinases have also been identified by molecular cloning. The human Erk1 protein kinase has been shown to be widely expressed as a 44‐kDa protein in many tissues. The human Erk2 protein kinase is a 41‐kDa protein that is expressed ubiquitously. In contrast, a human Erk3‐related protein kinase has been found to be expressed at a high level only in heart muscle and brain. The loci of these MAP kinase genes are widely distributed within the human genome: erk2 at 22q11.2; erk1 at 16p11.2; and ek3‐related at 18q12‐21.

In the yeast Saccharomyces cerevisiae, five MAP kinase gene homologs have been described: smk1, mpk1, hog1, fus3, and kss1. Together, these kinases are a more diverse group than the human erks that have been identified. Thus the erks are likely to represent only one subgroup of a larger human MAP kinase gene family. A candidate for this extended family of MAP kinases is the c‐Jun NH2‐terminal kinase (Jnk), which binds to and phosphorylates the transcription factor c‐Jun at the activating sites Ser‐63 and Ser‐73. Evidence is presented here to demonstrate that Jnk is a distant relative of the MAP kinase group that is activated by dual phosphorylation at Tyr and Thr. © 1995 wiley‐Liss, Inc.
Interactions between Ras and Raf: Key regulatory proteins in cellular transformation
Authors: Marshall Mark
Published: February 2005

Ras proteins function during cell growth and development as essential, plasma membrane‐bound signaling proteins. Current evidence suggests that Ras is part of a signal transduction chain extending from extracellular signals to transcriptional regulation in the nucleus. Growth factor and cytokine activation of many tyrosine kinase and kinase‐linked receptors recruits many proteins to the plasma membrane including Ras‐specific guanine nucleotide releasing proteins (GNRP). Under the influence of a GNRP, Ras proteins bind GTP, resulting in activation of the Ras signal. The GTP‐bound form of Ras is capable of interacting directly with RasGAP, neurofibromin, and the Raf kinases. Although believed to be endowed with some signaling capacity, RasGAP and neurofibromin act primarily to negatively regulate Ras. Based upon genetic and biochemical studies in a variety of diverse organisms, the Raf kinases are considered the primary targets of Ras signaling. Activation of the Raf kinases is the first step in a cascade of multiple protein kinases, including Mek, Erk1, and Erk2.

We are attempting to understand structurally how activated Ras proteins interact specifically with Raf kinases to induce the downstream signals necessary for cell division. Using mutagenesis, peptide epitope scanning, and in vitro reconstitution of protein interactions, we have identified specific sites of association between the Ras‐GTP and c‐Raf‐1 proteins. The interaction between these contact points is essential for the plasma membrane localization of Raf, which ultimately leads to kinase activation. The formation of this protein complex is negatively regulated by protein kinase A (PKA) through phosphorylation of the c‐Raf‐1 N‐terminus. Phosphorylation of c‐Raf‐1 serine 43 is believed to cause an N‐terminal cap structure to cover the Ras docking site. © 1995 wiley‐Liss, Inc.
Dissection of the Torso signal transduction pathway in Drosophila
Authors: Perrimon Norbert , Lu Xiangyi , Hou Xianyu Steven , Hsu Jui‐Chou , Melnick Michael B. , Chou Tze Bin , Perkins Lizabeth A.
Published: February 2005

Cell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP‐binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D‐Raf and D‐Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane‐bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D‐Raf and D‐Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D‐Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D‐raf mutation that abolishes binding of Ras1 to D‐Raf. These findings indicate that full D‐Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1‐independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso. © 1995 wiley‐Liss, Inc.
Influence of grain type on the insecticidal efficacy of two diatomaceous earth formulations against Rhyzopertha dominica (F) (Coleoptera: Bostrychidae)
Authors: Kavallieratos Nickolas G , Athanassiou Christos G , Pashalidou Foteini G , Andris Nickolas S , Tomanović Željko
Published: February 2005

Laboratory bioassays were carried out in order to evaluate the effectiveness of two diatomaceous earth (DE) formulations, Insecto and SilicoSec, against adults of Rhyzopertha dominica (F) in eight different grain commodities. The adherence of the two DEs to each grain was also measured. The eight grains tested were wheat, whole barley, peeled barley, oats, rye, triticale, rice and maize. These commodities were treated with the DEs at three rates, 0.75, 1.0 and 1.5 g DE kg−1 grain. The mortality of R dominica adults was measured after 24 h, 48 h, 7 days and 14 days of exposure in the treated grains at 26 °C and 55% RH. After the 14‐day mortality count, all adults were removed and the treated grains retained under the same conditions for a further 60 days. The treated grains were subsequently examined for F1 progeny. Significant differences were recorded among the eight grain types as well as between the DE formulations tested. After 14 days of exposure, even at the lowest DE rate for both formulations, adult mortality was high (>90%) in wheat and triticale. In contrast, adult mortality was significantly lower in peeled barley. Increasing the rate improved the efficacy of the DEs significantly in only some of the grains tested. Reproductive capacity in all the treated grains was significantly suppressed when compared with untreated grains. Generally, more F1 individuals of R dominica were noted in the treated peeled barley than in the other commodities. Significant differences in the percentage of DE retention were noted among the eight grains. The highest retention level was noted in rice (>87%) and the lowest in maize (<6%). However, the degree of DE adherence to a given grain was not always indicative of the effectiveness of DE against R dominica. Copyright © 2005 Society of Chemical Industry
Computed structure and energetics of La@C60
Authors: Slanina Zdeněk , Lee Shyi‐Long , Adamowicz Ludwik , Filip Uhlík  , Nagase Shigeru
Published: February 2005

Computations are carried out using density functional theory for the endohedral system La@C60, i.e., the very first metallofullerene observed in gas phase (although not yet isolated in a solid form). The computations offer data on the molecular and electronic structure of the species. In particular, it is shown that the encapsulated atom is not located in the cage center, but is shifted toward the wall. There is a substantial charge transfer from the metal to the cage, at some levels of theory amounting roughly to three electrons. It is shown that there is a relatively large energy stabilization upon encapsulation. However, the entropy term also influences the association equilibrium significantly, so that the standard Gibbs‐energy change depends strongly on temperature. The reasons why La@C60 could not be isolated yet are briefly discussed, as well as a possible relationship to superconductivity. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005
Binding of JAB1/CSN5 to MIF is mediated by the MPN domain but is independent of the JAMM motif
Authors: Burger-Kentischer Anke , Finkelmeier Doris , Thiele Michael , Schmucker Jürgen , Geiger Georg , Tovar Günter E.M. , Bernhagen Jürgen
Published: February 2005

Macrophage migration inhibitory factor (MIF) binds to c‐Jun activation domain binding protein‐1 (JAB1)/subunit 5 of COP9 signalosome (CSN5) and modulates cell signaling and the cell cycle through JAB1. The binding domain of JAB1 responsible for binding to MIF is unknown. We hypothesized that the conserved Mpr1p Pad1p N‐terminal (MPN) domain of JAB1 may mediate binding to MIF. In fact, yeast two hybrid (YTH) and in vitro translation/coimmunoprecipitation (CoIP) analysis showed that a core MPN domain, which did not cover the functional JAB1/MPN/Mov34 metalloenzyme (JAMM) deneddylase sequence, binds to MIF comparable to full‐length JAB1. YTH and pull‐down analysis in conjunction with nanobead affinity matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry demonstrated that MIF(50–65) and MPN are sufficient to mediate MIF–JAB1 interaction, respectively. Finally, endogenous CoIP of MIF–CSN6 complexes from mammalian cells demonstrated that MPN is responsible for MIF–JAB1 binding in vivo, and, as CSN6 does not contain a functional JAMM motif, confirmed that the interaction does not require JAMM.
A measure of near‐surface fluid motions that predicts air‐water gas transfer in a wide range of conditions
Authors: Turney Damon E. , Smith Walter C. , Banerjee Sanjoy
Published: February 2005

Air‐water gas transfer impacts many important biogeochemical processes, yet current understandings involve large uncertainty. This arises because the process depends on a complex interaction between molecular diffusion and fluid motions that has not been adequately characterized. Here we show the first experimental support for a mechanistic model that relates near‐surface motions to gas transfer coefficients over a range of wind conditions, including those leading to breaking wavelets. We find that the square root of the root mean square surface‐velocity divergence varies linearly with both the gas transfer coefficient, as predicted by theory, as well as with mean square surface slope. Besides advancing the understanding of the mechanisms governing air‐water gas transfer, these results suggest easy‐to‐measure parameters that could, with further investigation, provide gas transfer coefficients in field settings.
Interannual sea surface salinity changes in the western Pacific from 1954 to 2000 based on coral isotope analysis
Authors: Iijima Hiroko , Kayanne Hajime , Morimoto Maki , Abe Osamu
Published: February 2005

Changes in sea surface salinity (SSS) in the western Pacific warm pool (WPWP) provide information on the variability of the El Niño Southern Oscillation (ENSO) because SSS reflects the balance between evaporation and precipitation. We reconstructed the long‐term variability of SSS at the northwestern edge of the WPWP from oxygen isotope analysis of annual coral bands. The estimated SSS corresponded well with in situ observations, demonstrating that isotopic records from coral can be used as a paleo‐salinometer. Estimated SSS between 1954 and 2000 peaked during the El Niños of 1972–73, 1982–83, and 1997–98. These SSS peaks were caused by anomalously low precipitation accompanying the eastward shift in the WPWP.