bluuebunny/crossref_metadata_2025_split huggingface dataset:
LL95 monoclonal antibody mimics functional effects of ZP3 on mouse sperm: Evidence that the antigen recognized is not hexokinase
Authors: Leyton Lisette , Tomes Claudia , Saling Patricia 
Published: February 2005

AbstractIn the mouse, a 95 kD sperm protein has been identified as a putative receptor for the zona pellucida glycoprotein ZP3. The 95 kD sperm protein is a tyrosine kinase substrate, with phosphorylation on tyrosine stimulated upon zona pellucida binding. The latter finding is observed not only in live cells but also in isolated sperm membranes and in an electroeluted 95 kD protein. Stimulation of 95 kD protein tyrosine phosphorylation by zona pellucida is completely abolished by tyrosine kinase inhibitors, which effectively inhibit the sperm acrosome reaction. Since receptor oligomerization by ZP3 is essential for acrosome reaction triggering, we hypothesized that application of an external crosslinking agent will lead to the acrosome reaction, even in the absence of natural ligand ZP3. Here, we report the generation of a mouse monoclonal antibody (mAb) raised against the 95 kD protein. This antibody, termed LL95, mimics the bioactivities of ZP3 in inhibiting sperm‐zona binding and inducing the acrosome reaction. The latter depends on receptor oligomerization. Immunolocalization revealed that the LL95 antigen is restricted to the head surface in the acrosomal region of live sperm. Thus, LL95 fulfills several criteria predicted for an antibody that recognizes a sperm receptor for the zona pellucida. Recently, it was reported that the amino acid sequence of the 95 kD protein we described corresponds to a mouse hepatoma hexokinase (Kalab et al., 1994: J Biol Chem 269:3810–3817). Although both hexokinase and LL95 antigen migrate at 95 kD in nonreducing gels, we show here that LL95 does not recognize hexokinase. Identification of different proteins is clear where hexokinase is a 116 kD protein and LL95 recognizes sperm proteins of 110 and 130 kD. Moreover, mAb anti‐phosphotyrosine immunoprecipitates LL95 antigen under conditions where hexokinase is absent. Use of anti‐hexokinase antibodies in gamete interaction assays failed to demonstrate any effect on either spermzona binding or acrosome reaction triggering. Finally, anti‐hexokinase antibodies bind to a sperm tail antigen, thus direct involvement of hexokinase in gamete interaction seems improbable. © 1995 wiley‐Liss, Inc.
Distribution of RNase MRP RNA during Xenopus laevis oogenesis
Authors: Davis Alison F. , Jeong‐Yu Sunjoo , Clayton David A. 
Published: February 2005

AbstractRNase MRP is a ribonucleoprotein endoribonuclease found predominantly in nucleoli, but which has been associated with mitochondria and mitochondrial RNA processing. In order to analyze the intracellular localization of specific RNA components of ribonucleoproteins of this type, a whole‐mount method for in situ hybridization in Xenopus laevis oocytes was employed. Results with specific probes (for both mitochondrial and nonmitochondrial RNAs) indicate that this procedure is generally effective for the detection of a variety of nucleic acids that reside in different cellular compartments. Probes used to detect the endogenous RNA component of RNase MRP (MRP RNA) during X. laevis oogenesis revealed a continuous nuclear signal as well as a possible dual localization of MRP RNA in nucleoli and mitochondria at developmental stages temporally consistent with both ribosomal and mitochondrial biogenesis. Genomic DNA encoding MRP RNA was injected into the nuclei of stage VI oocytes and correctly transcribed. The in vivo–transcribed RNA was properly assembled with at least some of its cognate proteins as demonstrated by immunoprecipitation with specific autoantiserum. In addition, detectable levels of the RNA were exported to the cytoplasm. This whole‐mount procedure has permitted us to identify MRP RNA in situ at different developmental time points as well as during transcription of the injected gene, and suggests differential localization of MRP RNA during oogenesis consistent with its proposed function in both mitochondria and nucleoli. © 1995 wiley‐Liss, Inc.
Expression of serum albumin and of alphafetoprotein in murine normal and neoplastic primitive embryonic structures
Authors: Trojan J. , Naval X. , Johnson T. , Lafarge‐Frayssinet1 C. , Hajeri‐Germond M. , Farges O. , Pan Y. , Uriel J. , Abramasky O. , Ilan J. U. , Ilan J. 
Published: February 2005

AbstractAlphafetoprotein (AFP), a major serum protein synthesized during the embryo‐fetal and postnatal period (in the yolk sac, then in the liver), is also an oncoprotein. The intracellular presence of AFP and of serum albumin (SA) in normal and neoplastic neural crest and neural tube derivatives was previously demonstrated. In this work we have studied the comparative expression of AFP and SA in primitive neuroectoblastic structures of mouse embryos (6 and 7 days “post coitum”) and mouse teratocarcinomas (derived from the PCC4 cell line). Using immunofluorescence technique, antibodies to SA gave a positive reaction in embryos of 7 days, while AFP was not detected during this period. By mRNA in situ hybridization, SA mRNA gave a strong signal in both 6 and 7 day embryos, whereas AFP mRNA gave a weak signal only in 7‐day embryos. The distribution of SA and AFP and their mRNAs was investigated in primitive neuroectoblastic structures of the teratocarcinomas by in situ hybridization and immunostaining. Only SA protein was detectable by immunostaining. SA mRNA gave a strong signal in differentiating structures as well as in undifferentiated cell clusters. AFP mRNA was observed only in differentiating structures. Dot‐blot hybridization indicated that the level of SA transcripts was at least 6‐fold higher than that of AFP transcripts in the teratocarcinomas investigated. In teratocarcinoma‐bearing mice injected intraperitoneally with 125I‐radiolabeled SA and AFP, significant accumulations of both SA and AFP were demonstrated in the tumors, SA being about 3‐fold higher than that of AFP after normalization to quantity of uptake in liver. External in vivo photoscanning confirmed this relationship of accumulated radiolabeled proteins. The last observation could be useful in vivo for diagnosis of teratocarcinoma. We conclude that the expression of SA relative to AFP and the external cellular uptake of SA relative to AFP are similar in normal embryonic developing tissues and in the corresponding morphologically neoplastic tissues of the teratocarcinomas. The same SA:AFP relationship constitutes an oncofetal marker of primitive neuroectoblastic structures. © 1995 wiley‐Liss, Inc.
Identification of a unique μ‐class glutathione S‐transferase in mouse spermatogenic cells
Authors: Fulcher Kerry D. , Welch Jeffery E. , Klapper David G. , O'Brien Deborah A. , Eddy E. M. 
Published: February 2005

AbstractThe fibrous sheath is a major cytoskeletal structure in the principal piece of the mammalian sperm flagellum. Two peptide sequences obtained from a tryptic digest of mouse fibrous sheath proteins exhibited high homology with μ‐class glutathione S‐transferases (GSTs). Using a DNA probe amplified from degenerate polymerase chain reaction (PCR) primers predicted from these two peptide sequences, a ∼ 1.1 kb cDNA clone for fibrous sheath component 2 (Fsc2) was isolated which had 84% nucleic acid and 89% amino acid sequence identity with a previously reported μ‐class human GST gene (hGSTM3; Campbell et al., 1990: J Biol Chem 265:4188–9193). Sequences corresponding to those of the two fibrous sheath peptides were present in the protein encoded by the Fsc2 cDNA. Northern analysis with the full length Fsc2 cDNA detected a ∼ 1.1 kb mRNA in 12 of 15 somatic tissues examined, as well as in testis and isolated spermatogenic cells. However, 5′(nt − 96 to 12) or 3′ (nt 637 to 808) Fsc2 probes, containing mostly noncoding sequences, detected a ∼ 1.1 kb mRNA abundant in testis and isolated spermatogenic cells, but absent or present at low levels in somatic tissues. Northern analysis with RNA from testes of mice of different postnatal ages and purified spermatogenic cell populations indicated that this transcript is first present during the meiotic phase of germ cell development. These results suggest that a previously unreported μ‐class GST gene (mGSTM5*) is expressed at a specific time during the development of spermatogenic cells in the mouse. Immunoblot analysis indicated that a μ‐class GST protein is associated with the fibrous sheath, suggesting that it becomes an integral part of the mouse sperm cytoskeleton. © 1995 wiley‐Liss, Inc.
Factors affecting the developmental competence of mouse oocytes grown in vitro: Oxygen concentration
Authors: Eppig John J. , Wigglesworth Karen 
Published: February 2005

AbstractThe purpose of this study was to assess the effects of oxygen concentration on the developmental competence of mouse oocytes grown in vitro because oxygen has been shown to affect the nuclear maturation of oocytes (Haidri et al., 1971: J Reprod Fertil 26:409–411) and preimplantation embryo development (Whitten, 1971: Adv Biosci 6:129–139). Oocyte—granulosa cell complexes were isolated from the preantral follicles of 12‐day‐old mice and cultured for 10 days in serum‐free medium equilibrated with 5, 10, 15, or 20% O2. Five percent CO2 was used for all groups. Oocytes from all groups were then matured and fertilized, and preimplantation embryos cultured using 5% O2. With increased oxygen tension, there were dramatic decreases in the percentage of (1) oocytes that survived in vitro culture, (2) surviving oocytes that could resume meiosis and undergo germinal vesicle breakdown (GVB), and then cleave to the two‐cell stage, and (3) two‐cell‐stage embryos that completed the blastocyst transition. For example, 42% of the oocytes grown in a 5% O2 atmosphere cleaved to the two‐cell stage compared with only 3% when the oocytes grew in an atmosphere of 20% O2. These dramatic effects of elevated oxygen were mitigated by either increased numbers of the oocyte‐granulosa cell complexes in the culture or increased concentration of oxygen late in the culture period, after the oocytes became surrounded by greater numbers of granulosa cells. When the culture period was extended for 4 days beyond the standard 10 day cultures used in the experiments described above, there was an increased occurrence of precocious GVB or failure of the somatic cells to maintain meiotic arrest. This was prevented by increased oxygen concentration. Nevertheless, extending the time of culture even in the presence of elevated oxygen failed to increase oocyte growth to the equivalent of in vivo‐grown oocytes or to improve the developmental competence of the in vitro‐grown oocytes. It was concluded that concentrations of O2 above 5% have a deleterious effect on oocyte development during the early stages of culture. However, increasing the concentration on O2 during the later stages, in conjunction with other treatments to promote normal granulosa cell development and function and their interaction with the oocyte, may be critical to promote normal oocyte development in vitro. © 1995 wiley‐Liss, Inc.
Guanine nucleotide exchange factors: Activators of Ras superfamily proteins
Authors: Overbeck Ashley F. , Brtva Teresa R. , Cox Adrienne D. , Graham Suzanne M. , Huff Shayne Y. , Khosravi‐Far Roya , Quilliam Lawrence A. , Solski Patricia A. , Der Channing J. 
Published: February 2005

AbstractMembers of the Ras superfamily of proteins function as regulated GDP/GTP switches that cycle between active GTP‐complexed and inactive GDP‐complexed states. Guanine nucleotide exchange factors (GEFs) stimulate formation of the GTP‐bound state, whereas GTPase activating proteins (GAPs) catalyze the formation of the GDP‐bound state. We describe three studies that evaluate the mechanism of action of GEFs for Ras (SOS1 and RasGRF/CDC25) or Ras‐related Rho (Dbl and Vav) proteins.Growth factor‐mediated activation of Ras is believed to be mediated by activation of Ras GEFs (CDC25/GRF and SOS1/2). Although the mechanisms of Ras GEF regulation are unclear, recent studies suggest that translocation of SOS1 to the plasma membrane, where Ras is located, might be responsible for Ras activation. Our observation that the addition of the Ras plasma membrane‐targeting sequence to the catalytic domains of CDC25 and SOS1 greatly enhanced their transforming and transactivation activities (10–50 fold and 5–10 fold, respectively) suggests that membrane translocation alone is sufficient to potentiate GEF activation of Ras.We have determined that two Ras‐related proteins, designated R‐Ras and R‐Ras2/TC21, can trigger the malignant transformation of NIH 3T3 cells via activation of the Ras signal transduction pathway. Furthermore, like Ras and R‐Ras, we observed that TC21 GTPase activity was stimulated by Ras GAPs. However, we observed that both SOS1 and CDC25 were activators of normal TC21, but not R‐Ras, transforming activities. Therefore, TC21, but not R‐Ras, may be activated by the same extracellular signaling events that activate Ras proteins.Dbl family proteins are believed to function as GEFs and activators of the Ras‐related Rho family of proteins. However, one Dbl family oncogene, designated Vav, has been reported to be a GEF for Ras proteins. Therefore we were interested in determining whether Dbl family oncogenes cause transformation by triggering the constitutive activation of Rho or Ras proteins. Our results suggest that Dbl oncogenes cause transformation via a Ras‐independent activation of MAP kinases and Rho family proteins. © 1995 wiley‐Liss, Inc.
Ras‐related proteins in signal transduction and growth control
Authors: McCormick Frank 
Published: February 2005

AbstractRas proteins are members of a superfamily of small GTPases that are involved in many aspects of cell growth control. The ras p21 protooncogene products, H‐ras, K‐ras, and N‐ras, transmit signals from growth factor receptors to a cascade of protein kinases that begins with the Raf protooncogene product, and leads to alterations in transcription factors and cell cycle proteins in the nucleus. This cascade is controlled at several points: Ras p21 proteins are regulated by GAPs and by exchange factors, whose activities are altered by growth factor receptor activation (Boguski and McCormick, 1993: Nature 366:643–654). Transmission of signals from Ras to Raf is regulated by the Ras‐related protein Rap1 (a protein capable of reverting cell transformation) and by cAMP. Other aspects of Ras p21 regulation will be discussed, including the existence of RasGDl proteins that inhibit GDP dissociation from Ras, and may thus regulate the level of active Ras in the cell.The role of Ras in activation of Raf kinase appears to be limited to the recruitment of Raf to the plasma membrane, at which time Raf becomes stably modified to render it active (Leevers et al., 1994: Nature 369:411–414; Stokoe et al., 1994: Science 264:1463–1467). The nature of these modifications is unclear. Raf in the plasma membrane becomes associated with insoluble structural cell components that may be part of the activation. Furthermore, Raf is associated with proteins of the 14‐3‐3 family that appear necessary for kinase activation. The 14‐3‐3 proteins interact with all three conserved regions of Raf, including the kinase domain.In addition to Raf, Ras proteins interact with two known classes of proteins in a manner consistent with effector functions: these are the GAPs and regulators of the Ras‐related protein Ral referred to as RalGDS. These biochemical data suggest that other functional pathways are regulated by Ras, including, perhaps, pathways involved in regulating cell shape and motility.The protein R‐Ras p21 is about 50% identical to the Ras p21 protooncogene product. This protein is incapable of transforming cells, even though it interacts with Raf and other putative Ras effectors (Fernandez‐Sarabia and Bischoff, 1993: Nature 366:274–275). On the other hand, it has recently been shown that R‐Ras binds to the protooncogene product Bcl‐2, a protein that transforms B cells by blocking apoptosis. R‐Ras is regulated by the same GAP molecules as H‐Ras and the other Ras protooncogene products, and may therefore be activated in a manner co‐ordinate with these growth‐promoting proteins. The possible connection between R‐Ras and apoptosis will be discussed. © 1995 wiley‐Liss, Inc.
Synthesis and characterization of proton‐conducting copolyimides bearing pendant sulfonic acid groups
Authors: Yin Yan , Yamada Otoo , Suto Yoshiki , Mishima Takashi , Tanaka Kazuhiro , Kita Hidetoshi , Okamoto Ken‐ichi 
Published: February 2005

AbstractA series of sulfonated copolyimides (co‐SPIs) bearing pendant sulfonic acid groups were synthesized from 1,4,5,8‐naphthalenetetracarboxylic dianhydride (NTDA), bis(3‐sulfopropoxy) benzidines (BSPBs), and common nonsulfonated diamines via statistical or sequenced polycondensation reactions. Membranes were prepared by casting their m‐cresol solutions. The co‐SPI membrane had a microphase‐separated structure composed of hydrophilic and hydrophobic domains, but the connecting behavior of hydrophilic domains was different from that of the homo‐SPIs. The co‐SPI membranes displayed clear anisotropic membrane swelling in water with negligibly small dimensional changes in the plane direction of the membrane. With water uptake values of 39–94 wt %, they showed dimensional changes in membrane thickness of about 0.11–0.58, which were much lower than those of homo‐SPIs. The proton conductivity σ values of co‐SPI membranes with ion exchange capacity values ranging from 1.95–2.32 meq/g increased sigmoidally with increasing relative humidity. They displayed σ values of 0.05–0.16 S/cm at 50 °C in liquid water. Increasing temperature up to 120 °C resulted in further increase in proton conductivity. The co‐SPI membranes showed relatively good conductivity stability during the aging treatment in water at 100 °C for 300 h. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1545–1553, 2005
Dependence of the cyclization behavior of multifunctional network molecules on molecular size
Authors: Faliagas A. C. 
Published: February 2005

AbstractThe tendency for cyclization of nonlinear network molecules is quantitatively expressed by the cyclization probability, defined as the ratio of cyclization to total reaction rate. In contrast to branching, the rate of cyclization depends on the configurational statistical mechanics of segments joining functional groups. Direct integration of the joint configurational probability density in the subspace of encounters yields the distribution of the number of configurationally formed intramolecular functional group encounters (nearest neighbors). Statistically independent network segments are assumed to obey the Gaussian statistics. A recursive relationship for the distribution of chain lengths is developed, and it is shown that for large molecular sizes this relationship tends to a limiting distribution. Corresponding average and standard deviation follow power law dependence on degree of polymerization (DP). With these results, cyclization probabilities are explicitly expressed as functions of DP. When segmental diffusion is the rate controlling factor, cyclization is similar to short range (or primary) cyclization. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 861–870, 2005
The carboxyterminal processing protease of D1 protein: expression, purification and enzymology of the recombinant and native spinach proteins
Authors: Fabbri Bradon J , Duff Stephen MG , Remsen Edward E , Sophia Chen Yun‐Chia , Anderson John C , CaJacob Claire A 
Published: February 2005

AbstractThe carboxyterminal processing protease of D1 protein (CtpA) is predicted to be an excellent target for a general broad‐spectrum herbicide. The gene for spinach CtpA has been expressed in Escherichia coli. The expressed protein that was found mainly in inclusion bodies has been purified and refolded on a nickel‐chelate column. Active recombinant CtpA was recovered. Two assays for CtpA activity were developed, a medium‐throughput HPLC assay using a fluorescent substrate and a high‐throughput assay based on fluorescence polarization capable of application in a high‐throughput 96‐well plate format. This high‐throughput assay was developed to screen chemistry for CtpA inhibitors. Native spinach CtpA was partially purified and the native and recombinant enzymes were compared kinetically for their Km and Vmax values using different peptide substrates. Native CtpA partially purified from spinach was shown to have similar kinetic properties to recombinant CtpA. Antibodies developed against the recombinant protein were used to estimate the in planta abundance of the native enzyme in spinach. Since only a small proportion of the recombinant protein is refolded during isolation and it appears that only a small proportion of this enzyme is active, size‐exclusion chromatography and light scattering experiments were performed on rCtpA in order to gain insight into its structure and the reasons why most of the protein is not active. The use of rCtpA to screen for herbicidal compounds and the more general question of how good a herbicide target the enzyme is are discussed. Copyright © 2005 Society of Chemical Industry
Noradrenergic and dopaminergic effects of (+)‐amphetamine‐like stimulants in the baboon Papio anubis
Authors: Alexander Mohab , Rothman Richard B. , Baumann Michael H. , Endres Christopher J. , Brašić James Robert , Wong Dean F. 
Published: February 2005

Abstract(+)‐Amphetamine, (±)‐ephedrine, and phentermine are commonly used appetite suppressants that release monoamines from nerve cells by acting as substrates for biogenic amine transporters. One key difference among the three drugs is their selectivity for norepinephrine (NE) release vs. dopamine (DA) release. The NE/DA selectivity ratios for these drugs as determined in vitro [(EC50 NE−1)/(EC50 DA−1)] are (±)‐ephedrine (18.6) > phentermine (6.7) > (+)‐amphetamine (3.5). The in vitro data suggest that when administered in vivo, these stimulants might differ in their ability to release DA from nerve terminals in the brain. To test this hypothesis, noradrenergic effects (i.e., plasma NE) and dopaminergic effects (i.e., central DA release) were assessed when each drug was administered intravenously (1.5 mg/kg) to anesthetized baboons. Central DA release was determined via positron emission tomography using the method of [11C]raclopride displacement. In the present investigation, high doses of these stimulants increased plasma NE and DA in parallel, but only (+)‐amphetamine released central DA from neurons and decreased plasma prolactin. None of the drugs altered plasma amine metabolite levels, indicating no inhibition of monoamine oxidase activity at the administered doses. Plasma drug levels measured in baboons were higher than those measured in human patients taking prescribed doses of the drugs. Viewed collectively, the present data indicate that typical clinical doses of phentermine and (±)‐ephedrine may not release central DA in humans, a hypothesis that should ultimately be tested in controlled clinical studies. Synapse 56:94–99, 2005. Published 2005 Wiley‐Liss, Inc.