bluuebunny/crossref_metadata_2025 huggingface dataset:
5‐aza deoxycytidine‐induced inhibition of differentiation of spermatogonia into spermatocytes in the mouse
Authors: Raman Rajiva , Narayan Gopeshwar
Published: February 2005

In order to explore the significance of DNA methylation in proliferation and differentiation of germ cells in testis, 5‐aza,2′‐deoxycytidine (5‐azaCdR), a hypomethylating agent, was administered in vivo to neonatal mice having only spermatogonial (premeiotic) cells. End‐labeling of the Mspl, Hpall, and Hhal digested DNA revealed considerable loss of methylation following the treatment. Cellular and histological preparations of the testis showed complete inhibition of differentiation into spermatocytic stage. Analysis of protein synthesis in the treated and control testis by growing the cells in 35S‐Methionine medium and resolving the lysate by SDS‐PAGE revealed that the programme of expression of at least 5 polypeptides (35.0, 31.5, 27.0, 22.5, and 18.0 KD) was altered as a result of 5‐azaCdR incorporation. It appears that DNA methylation plays a critical role in the differentiation of gonia into primary spermatocytes. © 1995 wiley‐Liss, Inc.
Initiation of transcription and nucleologenesis in equine embryos
Authors: Brinsko S. P. , Ball B. A. , Ignotz G. G. , Thomas P. G. A. , Currie W. B. , Ellington J. E.
Published: February 2005

The time of activation of the embryonic genome (maternal‐embryonic transition) in equine embryos was investigated by assessing incorporation of3H‐uridine and nucleolar development. In Experiment 1, embryos were recovered from the oviduct (n = 15) and the uterus (n = 3). Recovered embryos were assessed for morphologic development and quality score. Recovered embryos with less than 8 cells (two cells, n = 4; four cells, n = 5; five cells, n = 2) were incubated with3H‐uridine (560 μCi/ml) for 10 hr, while eight‐cell embryos (n = 2), morulae (n = 2), and blastocysts (n = 3) were incubated with 280 μCi/ml for 0.5–1 hr. At the end of incubation, embryos were washed twice in PBS with 10% FBS and incubated for 30 min with 2.5 mg/ml of unlabelled uridine. Embryos were spread onto glass slides, dipped into emulsion, and exposed for 8 d, then developed and counter‐stained with Giemsa and propidium iodide. Embryos at the blastocyst, morula, eight‐cell, and five‐cell stages incorporated3H‐uridine into their cell nuclei as detected by autoradiography. In a second experiment, nucleologenesis in equine embryos was examined by transmission electron microscopy. Nucleoli or nucleolar precursors were found in 12 of 23 embryos examined. Most embryos in the four‐ to six‐cell stage (n = 7) had nucleolar precursor bodies (npb) consisting of homogeneous fibrillar structures. Two five‐ to six‐cell embryos also possessed reticulated nucleoli with both fibrillar and granular components as did all eight‐cell embryos (n = 3). Nucleoli in one morula and one blastocyst were reticulated with prominent granular components, fibrillar components, and apparent fibrillar centers. These results indicate that incorporation of3H‐uridine and the formation of functional nucleoli with typical fibrillar and granular components occurs between the four‐ to eight‐cell stage in equine embryos. © 1995 wiley‐Liss, Inc.
Mechanism of action of epidermal growth factor‐induced porcine oocyte maturation
Authors: Coskun Serdar , Lin Young C.
Published: February 2005

EGF has been reported to promote oocyte maturation in several species, although the mechanism of action is not yet known. The present study is designed to determine the pathway used by EGF to enhance porcine oocyte maturation. Oocytes were aspirated from 2–5 mm follicles and cultured with various treatments in Medium‐199 at 37°C, 100% relative humidity, and 5% CO2 for 48 hr for the maturation study and 3 hr for intracellular cAMP measurement. Although treatment with 100 IU/ml hCG stimulated both intracellular cAMP formation and oocyte maturation, 10 ng/ml EGF stimulated oocyte maturation only. Dibutyryl cAMP (dbcAMP) inhibited oocyte maturation at 10−5, 10−4, and 10−3 M concentrations in the control medium. However, in the presence of 10 ng/ml EGF, dbcAMP inhibited oocyte maturation only at a concentration of 10−3 M. Increasing concentrations of EGF (i.e., 25 and 50 ng/ml) were ineffective in overcoming the inhibitory effect of dbcAMP at 10−3 M. In contrast, EGF reversed the decreased maturation rate caused by transforming growth factor‐β. Phorbol myristate acetate (PMA), a tumor‐promoting phorbol ester, enhanced the spontaneous maturation rate; 4α‐phorbol dideconoate, an inactive phorbol ester, did not show this effect. PMA‐ and EGF‐stimulated porcine oocyte maturation is reversed by calphostin‐C, a PKC inhibitor. In conclusion, EGF's promotional activity on porcine oocyte maturation is independent of the cAMP pathway and probably mediated by the PKC pathway. © 1995 wiley‐Liss, Inc.
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

In 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

RNase 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

Alphafetoprotein (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

The 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

The 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

Members 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

Ras 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

A 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

The 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

The 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

(+)‐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.
Prefrontal cortical cell firing during maintenance, extinction, and reinstatement of goal‐directed behavior for natural reward
Authors: Peters Yvette M. , O'Donnell Patricio , Carelli Regina M.
Published: February 2005

The prefrontal cortex (PFC) is important for higher cognitive functioning and the processing of reward‐related information. Here, electrophysiological recording procedures were used to examine cell firing in the PFC in rats (n = 12) during water reinforcement sessions consisting of three phases. In phase one (maintenance), animals pressed a lever (fixed ratio 1) for water reinforcement (0.05 ml/press) paired with an auditory stimulus. Of 62 neurons recorded during maintenance, 39 (63%) exhibited one of three types of patterned discharges relative to the reinforced response for water. Specifically, PFC neurons exhibited increases in firing rate within seconds preceding the response (type PR; n = 9 cells) or increases (type RFe; n = 16 cells) or decreases (type RFi; n = 14 cells) in firing rate immediately following response completion. The remaining neurons did not alter their firing profiles relative to the reinforced response (type nonphasic cells; n = 23 cells). In phase two (extinction), lever press responses had no programmed consequences (i.e., water reinforcement and the auditory stimulus were not presented). After 30 min of no responding, phase three (reinstatement) began, during which each lever press response was again associated with water reinforcement paired with the stimulus. Results indicate differential effects of extinction/reinstatement on cell firing rates and patterns dependent on cell type. These findings are discussed with respect to the adaptive nature of PFC activity during goal‐directed behaviors for “natural” rewards, and are considered relative to prior studies that examined nucleus accumbens cell firing during a similar behavioral task. Synapse 56:74–83, 2005. © 2005 Wiley‐Liss, Inc.
Neuropathology of the cerebral cortex observed in a range of animal models of prenatal cocaine exposure may reflect alterations in genes involved in the Wnt and cadherin systems
Authors: Novikova Svetlana I. , He Fang , Bai Jie , Lidow Michael S.
Published: February 2005

Several recent reports show that the cerebral cortex in humans and animals with altered expressions of Wnt/cadherin network‐associate molecules display cytoarchitectural abnormalities reminiscent of cortical dysplasias seen in some (mouse‐, rat‐, and monkey‐based) animal models of prenatal cocaine exposure. Therefore, we employed oligo microarrays followed by real‐time RT‐PCR to compare expressions of genes involved in Wnt and cadherin systems in the cerebral wall of 18‐day‐old (E18) fetuses from cocaine‐treated (20 mg/kg cocaine, s.c., b.i.d., E8–18) and drug‐naive (saline, s.c.) mice. The pregnant mice chronically treated with cocaine in the above‐described manner represent one of the animal models producing offspring with widespread cortical dysplasias. Out of more than 150 relevant genes in the arrays, 32 were upregulated and 9 were downregulated in cocaine‐exposed fetuses. The majority of these genes (30 out of 41) were similarly affected in the frontal and occipital regions of the cerebral wall. We also used Western immunoblotting to examine the ability of cocaine to regulate the protein levels of β‐catenin, the key functional component of both Wnt and cadherin systems. While the total cell levels of β‐catenin were increased throughout the cerebral wall of cocaine‐exposed fetuses, its nuclear (gene‐transcription driving) levels remained unaltered. This suggests a transcription‐unrelated role for cocaine‐induced upregulation of this protein. Overall, our findings point to an intriguing possibility that that cerebral cortical dysplasias observed in several animal models of prenatal cocaine exposure may be at least in part related to alterations in the Wnt/cadherin molecular network. Synapse 56:105–116, 2005. © 2005 Wiley‐Liss, Inc.
Ultra‐high resolution imaging of DNA and nucleosomes using non‐contact atomic force microscopy
Authors: Davies Emma , Teng Kar Seng , Conlan Robert Steven , Wilks Stephen Patrick
Published: February 2005

Visualisation of nano‐scale biomolecules aids understanding and development in molecular biology and nanotechnology. Detailed structure of nucleosomes adsorbed to mica has been captured in the absence of chemical‐anchoring techniques, demonstrating the usefulness of non‐contact atomic force microscopy (NC‐AFM) for ultra‐high resolution biomolecular imaging. NC‐AFM offers significant advantages in terms of resolution, speed and ease of sample preparation when compared to techniques such as cryo‐electron microscopy and X‐ray crystallography. In the absence of chemical modification, detailed structure of DNA deposited on a gold substrate was observed for the first time using NC‐AFM, opening up possibilities for investigating the electrical properties of unmodified DNA.
Interannual variability of the 10–25‐ and 30–60‐day variation over the South China Sea during boreal summer
Authors: Kajikawa Yoshiyuki , Yasunari Tetsuzo
Published: February 2005

The interannual variability (IAV) of the intraseasonal variation (ISV) over the South China Sea (SCS) was investigated, focusing on the relationship between the 10–25‐ and 30–60‐day variation. Both ISV activities over the SCS had larger IAV than those of the Bay of Bengal and Arabian Sea. The 10–25‐ (30–60‐) day variation was relatively active when the 30–60‐ (10–25‐) day variation was suppressed in June, July and September. The IAV of the ISV during early summer (June–July) was related to the IAV of the SCS monsoon onset. A late (early) onset was accompanied by active 10–25‐ (30–60‐) day variation. The IAV of the ISV could be divided into early and late summer (September), because there was little persistence between these two periods. Moreover, large‐scale circulation anomalies associated with the IAV had different spatial structures in these two periods.
Effect of plastic‐viscous layering and strain softening on mode selection during lithospheric extension
Authors: Huismans Ritske S. , Buiter Susanne J. H. , Beaumont Christopher
Published: February 2005

Factors controlling the selection of deformation modes during continental extension are investigated using analytical and numerical methods. We view the lithosphere as a laminate and examine a simple system with a uniform plastic layer overlying a uniform linear viscous layer. The rate of energy dissipation is analyzed for pure shear (PS), symmetric plug (SP), and asymmetric plug (AP) extension modes, and the analysis reveals that the primary control is the relative rate of dissipation in the two layers. A basic difference is that the plastic layer yield strength is independent of the strain rate, whereas the viscous stress depends on strain rate; therefore dissipation scales linearly and quadratically with extension velocity for these respective layers. When other parameters, e.g., extension velocity, and properties are held constant, minimum dissipation predicts that the modes AP, SP, and PS will be selected in this order with increasing viscosity of the lower layer. Transition viscosities between modes, ηT1 and ηT2 are 4 × 1021 Pa s and 8 × 1022 Pa s, respectively, for our parameters values. Numerical models confirm the analysis results, inferred mode controls, and order of mode selection when strain softening of the plastic layer occurs during extension. Implications for lithosphere that acts as a bonded plastic/viscous laminate include the following (1) asymmetric extension (AP mode) is preferred when the extension rate and/or effective viscosity is low or the viscous region temperature is high, (2) symmetric (SP mode) extension is preferred for intermediate combinations of parameters, and (3) overall pure shear (PS mode) may occur for opposite end‐member parameter combinations.
Relationship between depth and age in the North Pacific Ocean
Authors: Hillier J. K. , Watts A. B.
Published: February 2005

The North Pacific contains active mid‐oceanic ridges and the oldest, Jurassic (166.8 ± 4 Ma), drilled oceanic crust. Its bathymetry is therefore critical to studies of the applicability of thermal contraction models (e.g., the infinite half‐space and cooling plate) to the subsidence of seafloor with crustal age. The bathymetry, however, contains seamounts and oceanic islands (e.g., Mid‐Pacific Mountains), oceanic plateaus (e.g., Hess, Magellan, and Shatsky), and midplate topographic swells (e.g., Hawaii), which are unrelated to the current plate‐scale thermal state of the oceanic lithosphere. We use here a regional‐residual separation algorithm called MiMIC to remove these features and to isolate the depths associated with the subsidence of North Pacific oceanic crust. These depths, z (m), increase with time, t (Ma), as z = 3010 + 307 until 85 Ma. For greater ages the depths “flatten” and asymptotically approach ∼6.1 km and are well described by z = 6120 − 3010 exp(−0.026t). The flattening is not “abrupt” as recently described in z‐t curves produced using the mean, median, and mode. As a result, the depths of both young and old seafloor are fit well (mean difference between and observed and calculated depths of 75 ± 54 m 1σ) by a single cooling plate model. Using a thermal conductivity, k, of 3.138 mW m−2 as previous studies, however, gives a plate of similar thickness (i.e., thermal thickness, L, of ∼115 km) but one which is unreasonably hot (i.e., temperature at the base of the plate, Tb, of 1522 °C) and inexpansive (i.e., coefficient of thermal expansion, α, of 2.57×10−5 °C−1). More reasonable values (i.e., Tb = 1363°C, k = 3.371 W m−1 °C−1, α = 2.77×10−5 °C−1, and L = 120 km) are obtained if the crustal thickness is used to constrain Tb and a certain amount of the surface heat flow is allowed to be radiogenically generated within the oceanic lithosphere.
Modeling seasonal vegetation variation and its validation against Moderate Resolution Imaging Spectroradiometer (MODIS) observations over North America
Authors: Kim Yeonjoo , Wang Guiling
Published: February 2005

Seasonal variability of vegetation, determined by plant phenology, impacts the seasonality of surface and atmospheric water cycles as well as the seasonality of surface energy budget. At the same time, leaf seasonal variations respond to both cumulative and concurrent hydrometeorological conditions. In order to account for this vegetation feedback at the seasonal timescale, a predictive phenology scheme for various plant functional types is developed on the basis of previous studies, and a methodology for crop simulations is proposed and implanted to supplement this phenology scheme. The phenology scheme is then incorporated into the Community Land Model (CLM). The geographic focus of this study is on the United States where the need for seasonal prediction is urgent and vegetation seasonal characteristics have been shown to significantly influence summer precipitation and temperature. Comparison of the model simulation with Moderate Resolution Imaging Spectroradiometer (MODIS)‐derived leaf area index data indicates that our model reproduces the observed vegetation seasonality reasonably well. Subsequent experiments demonstrate the interannual variability of vegetation phenology and its impact on surface water and energy budgets using the 1988 drought and 1993 flood in the U.S. Midwest as examples.
A Dopamine D4 Receptor Antagonist Attenuates Ischemia-Induced Neuronal Cell Damage via Upregulation of Neuronal Apoptosis Inhibitory Protein
Authors: Okada Yoshinori , Sakai Harumi , Kohiki Eri , Suga Etsuko , Yanagisawa Yoshiko , Tanaka Kazunori , Hadano Shinji , Osuga Hitoshi , Ikeda Joh-E
Published: February 2005

Neuronal apoptosis inhibitory protein (NAIP/BIRC1), the inhibitor of apoptosis protein (IAP) family member, suppresses neuronal cell death induced by a variety of insults, including cell death from ischemia and stroke. The goal of the present study was to develop an efficient method for identification of compounds with the ability to upregulate endogenous NAIP and to determine the effects on these compounds on the cellular response to ischemia. A novel NAIP-enzyme-linked immunosorbent assay (ELISA)-based in vitro drug-screening system is established. Use of this system identified an antagonist of dopamine D4 receptor, termed L-745,870, with a potent NAIP upregulatory effect. L-745,870-mediated NAIP upregulation in neuronal and nonneuronal cultured cells resulted in decreased vulnerability to oxidative stress-induced apoptosis. Reducing NAIP expression via RNA interference techniques resulted in prevention of L-745,870-mediated protection from oxidative stress. Further, systemic administration of L-745,870 attenuated ischemia-induced damage of the hippocampal CA1 neurons and upregulated NAIP expression in the rescued hippocampal CA1 neurons in a gerbil model. These data suggest that the NAIP upregulating compound, L-745,870, has therapeutic potential in acute ischemic disorders and that our NAIP-ELISA-based drug screening may facilitate the discovery of novel neuroprotective compounds.
Angiotensin II AT1 Receptor Blockade Abolishes Brain Microvascular Inflammation and Heat Shock Protein Responses in Hypertensive Rats
Authors: Zhou Jin , Ando Hiromichi , Macova Miroslava , Dou Jingtao , Saavedra Juan M
Published: February 2005

Endothelial dysfunction and inflammation enhance vulnerability to hypertensive brain damage. To explore the participation of Angiotensin II (Ang II) in the mechanism of vulnerability to cerebral ischemia during hypertension, we examined the expression of inflammatory factors and the heat shock protein (HSP) response in cerebral microvessels from spontaneously hypertensive rats and their normotensive controls, Wistar Kyoto rats. We treated animals with vehicle or the Ang II AT1 receptor antagonist candesartan, 0.3 mg/kg/day, via subcutaneously implanted osmotic minipumps for 4 weeks. Spontaneously hypertensive rats expressed higher Angiotensin II AT1 receptor protein and mRNA than normotensive controls. Candesartan decreased the macrophage infiltration and reversed the enhanced tumor necrosis factor-α and interleukin-1β mRNA and nuclear factor-κB in microvessels in hypertensive rats. The transcription of many HSP family genes, including HSP60, HSP70 and HSP90, and heat shock factor-1 was higher in hypertensive rats and was downregulated by AT1 receptor blockade. Our results suggest a proinflammatory action of Ang II through AT1 receptor stimulation in cerebral microvessels during hypertension, and very potent antiinflammatory effects of the Ang II AT1 receptor antagonist. These compounds might be considered as potential therapeutic agents against ischemic and inflammatory diseases of the brain.
Cerebral Oxygenation during Postasphyxial Seizures in Near-Term Fetal Sheep
Authors: Gonzalez Hernan , Hunter Christian J , Bennet Laura , Power Gordon G , Gunn Alistair J
Published: February 2005

After exposure to asphyxia, infants may develop both prolonged, clinically evident seizures and shorter, clinically silent seizures; however, their effect on cerebral tissue oxygenation is unclear. We therefore examined the hypothesis that the increase in oxygen delivery during postasphyxial seizures might be insufficient to meet the needs of increased metabolism, thus causing a fall in tissue oxygenation, in unanesthetized near-term fetal sheep in utero (gestational age 125 ± 1 days). Fetuses were administered an infusion of the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine, followed by 10 mins of asphyxia induced by complete umbilical cord occlusion. The fetuses then recovered for 3 days. Sixty-one episodes of electrophysiologically defined seizures were identified in five fetuses. Tissue PO2 (tPO2) did not change significantly during short seizures (<3.5 mins), 5.2 ± 0.2 versus baseline 5.6 ± 0.1 mm Hg (NS), but fell to 2.2 ± 0.2 mm Hg during seizures lasting more than 3.5 mins ( P<0.001). During prolonged seizures, cortical blood flow did not begin to increase until tPO2 had begun to fall, and then rose more slowly than the increase in metabolism, with a widening of the brain to blood temperature gradient. In conclusion, in the immature brain, during prolonged, but not short seizures, there is a transient mismatch between cerebral blood flow and metabolism leading to significant cerebral deoxygenation.
Human mesenchymal stem cells in rodent whole-embryo culture are reprogrammed to contribute to kidney tissues
Authors: Yokoo Takashi , Ohashi Toya , Shen Jin Song , Sakurai Ken , Miyazaki Yoichi , Utsunomiya Yasunori , Takahashi Masanori , Terada Yoshio , Eto Yoshikatsu , Kawamura Tetsuya , Osumi Noriko , Hosoya Tatsuo
Published: February 2005

The use of stem cells has enabled the successful generation of simple organs. However, anatomically complicated organs such as the kidney have proven more refractory to stem-cell-based regenerative techniques. Given the limits of allogenic organ transplantation, an ultimate therapeutic solution is to establish self-organs from autologous stem cells and transplant them as syngrafts back into donor patients. To this end, we have striven to establish an
in vitro
organ factory to build up complex organ structures from autologous adult stem cells by using the kidney as a target organ. Cultivation of human mesenchymal stem cells in growing rodent embryos enables their differentiation within a spatially and temporally appropriate developmental milieu, facilitating the first step of nephrogenesis. We show that a combination of whole-embryo culture, followed by organ culture, encourages exogenous human mesenchymal stem cells to differentiate and contribute to functional complex structures of the new kidney.
Amphetamine induces dopamine efflux through a dopamine transporter channel
Authors: Kahlig Kristopher M. , Binda Francesca , Khoshbouei Habibeh , Blakely Randy D. , McMahon Douglas G. , Javitch Jonathan A. , Galli Aurelio
Published: February 2005

Drugs of abuse, including cocaine, amphetamine (AMPH), and heroin, elevate extracellular dopamine (DA) levels in the brain, thereby altering the activity/plasticity of reward circuits and precipitating addiction. The physiological release of DA occurs through the calcium-dependent fusion of a synaptic vesicle with the plasma membrane. Extracellular DA is cleared by uptake through the Na
+
/Cl
-
-dependent DA transporter (DAT). In contrast, the substrate AMPH induces nonvesicular release of DA mediated by DAT. Extracellular AMPH is generally believed to trigger DA efflux through DAT by facilitating exchange for cytosolic DA. Here, in outside-out patches from heterologous cells stably expressing DAT or from dopaminergic neurons, by using ionic conditions in the patch pipette that mimic those produced by AMPH stimulation, we report that AMPH causes DAT-mediated DA efflux by two independent mechanisms: (
i
) a slow process consistent with an exchange mechanism and (
ii
) a process that results in rapid (millisecond) bursts of DA efflux through a channel-like mode of DAT. Because channel-like release of DA induced by AMPH is rapid and contains a large number of DA molecules, with a single burst of DA on par with a quantum of DA from exocytotic release of a vesicle, this burst mode of release may play a role in the synaptic actions and psychostimulant properties of AMPH and related compounds. Unlike AMPH, the endogenous substrate DA, when present on both sides of the plasma membrane, inhibits this channel-like activity, thereby suggesting that the DAT channel-like mode cannot accumulate DA against a concentration gradient.