Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes

Receptors for N-methyl-D-aspartate (NMDA) are involved in many plastic and pathological processes in the brain. Glycine has been reported to potentiate NMDA responses in neurons and in Xenopus oocytes injected with rat brain messenger RNA. Glycine is now shown to be absolutely required for activation of NMDA receptors in oocytes. In voltage-clamped oocytes, neither perfusion nor rapid pressure application of NMDA onto messenger RNA-injected oocytes caused a distinct ionic current without added glycine. When glycine was added, however, NMDA evoked large inward currents. The concentration of glycine required to produce a half-maximal response was 670 nanomolar, and the glycine dose-response curve extrapolated to zero in the absence of glycine. Several analogs of glycine could substitute for glycine, among which D-serine and D-alanine were the most effective. The observation that D-amino acids are effective will be important in developing drugs targeted at the glycine site.     

Expression of a cloned rat brain potassium channel in Xenopus oocytes

Potassium channels are ubiquitous membrane proteins with essential roles in nervous tissue, but little is known about the relation between their function and their molecular structure. A complementary DNA library was made from rat hippocampus, and a complementary DNA clone (RBK-1) was isolated. The predicted sequence of the 495-amino acid protein is homologous to potassium channel proteins encoded by the Shaker locus of Drosophila and differs by only three amino acids from the expected product of a mouse clone MBK-1. Messenger RNA transcribed from RBK-1 in vitro directed the expression of potassium channels when it was injected into Xenopus oocytes. The potassium current through the expressed channels resembles both the transient (or A) and the delayed rectifier currents reported in mammalian neurons and is sensitive to both 4-aminopyridine and tetraethylammonium.     

Blockade of N-methyl-D-aspartate receptors may protect against ischemic damage in the brain

In rats ischemia of the forebrain induced by a 30-minute occlusion of the carotid artery, followed by 120 minutes of arterial reperfusion, produced ischemic lesions of selectively vulnerable pyramidal cells in both hippocampi. Focal microinfusion into the dorsal hippocampus of 2-amino-7-phosphonoheptanoic acid, an antagonist of excitation at the N-methyl-D-aspartate-preferring receptor, before ischemia was induced protected against the development of ischemic damage. It is proposed that excitatory neurotransmission plays an important role in selective neuronal loss due to cerebral ischemia.     

Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes

Intracellular calcium (Ca2+) is a ubiquitous second messenger. Information is encoded in the magnitude, frequency, and spatial organization of changes in the concentration of cytosolic free Ca2+. Regenerative spiral waves of release of free Ca2+ were observed by confocal microscopy in Xenopus laevis oocytes expressing muscarinic acetylcholine receptor subtypes. This pattern of Ca2+ activity is characteristic of an intracellular milieu that behaves as a regenerative excitable medium. The minimal critical radius for propagation of focal Ca2+ waves (10.4 micrometers) and the effective diffusion constant for the excitation signal (2.3 x 10(-6) square centimeters per second) were estimated from measurements of velocity and curvature of circular wavefronts expanding from foci. By modeling Ca2+ release with cellular automata, the absolute refractory period for Ca2+ stores (4.7 seconds) was determined. Other phenomena expected of an excitable medium, such as wave propagation of undiminished amplitude and annihilation of colliding wavefronts, were observed.     

Aggregation of lysine-containing zeins into protein bodies in Xenopus oocytes

Zeins, the storage proteins of maize, are totally lacking in the essential amino acids lysine and tryptophan. Lysine codons and lysine- and tryptophan-encoding oligonucleotides were introduced at several positions into a 19-kilodalton zein complementary DNA by oligonucleotide-mediated mutagenesis. A 450-base pair open reading frame from a simian virus 40 (SV40) coat protein was also engineered into the zein coding region. Messenger RNAs for the modified zeins were synthesized in vitro with an SP6 RNA polymerase system and injected into Xenopus laevis oocytes. The modifications did not affect the translation, signal peptide cleavage, or stability of the zeins. The ability of the modified zeins to assemble into structures similar to maize protein bodies was assayed by two criteria: assembly into membrane-bound vesicles resistant to exogenously added protease, and ability to self-aggregate into dense structures. All of the modified zeins were membrane-bound; only the one containing a 17-kilodalton SV40 protein fragment was unable to aggregate. These findings suggest that it may be possible to create high-lysine corn by genetic engineering.     

Potassium salt microinjection into Xenopus oocytes mimics gonadotropin treatment

Gonadotropin stimulates protein synthesis and growth in ovarian oocytes. The hormone is also known to modify transfollicular K+ fluxes and is now shown to cause increased intraoocytic K+ activity (aK). The hormone's effect on aK was duplicated by microinjecting K+ salts into oocytes which were incubated in paraffin oil. This treatment mimicked the influence of gonadotropin on both the rate of protein synthesis and the synthesis of specific polypeptides. These findings suggest that gonadotropin-stimulated oocyte growth is attributable largely to the hormone's influence on transfollicular K+ fluxes. They support the hypothesis that the K+ flux and aK changes observed during cell activation are critical in causing subsequent increases in protein synthesis and growth.     

Collagen synthesis in Xenopus oocytes after injection of nuclear RNA of frog embryos

A messenger RNA fraction from polysomes of frog larvae or RNA preparations from isolated nuclei of developing frog embryos were injected into growing Xenopus laevis oocytes that were incubated with labeled proline. Column chromatography of protein hydrolyzates revealed labeled hydroxyproline after injection of the messenger RNA fraction and neurula nuclear RNA, indicating that the injected material had promoted collagen synthesis.     

Ras p21 as a potential mediator of insulin action in Xenopus oocytes

The oncogene protein product (p21) of the ras gene has been implicated in mediating the effects of a variety of growth factors and hormones. Microinjection of monoclonal antibody 6B7, which is directed against a synthetic peptide corresponding to a highly conserved region of p21 (amino acids 29 to 44) required for p21 function, specifically inhibited Xenopus oocyte maturation induced by incubation with insulin. The inhibition was dose-dependent and specific since (i) the same antibody had no effect on progesterone-induced maturation, (ii) immunoprecipitation and Western blotting indicated that the antibody recognized a single protein of molecular weight 21,000 in oocyte extracts, and (iii) inhibition was not observed with identical concentrations of normal immunoglobulin. Thus, p21 appears to be involved in mediating insulin-induced maturation of Xenopus oocytes. Furthermore, the mechanism may involve phosphorylation of p21, as p21 was found to be a substrate of the insulin receptor kinase.     

Nuclear factors in B lymphoma enhance splicing of mouse membrane-bound mu mRNA in Xenopus oocytes

Regulation of the synthesis of membrane-bound and secreted immunoglobulin mu heavy chains at the level of RNA processing is an important element for B cell development. The precursor mu RNA is either polyadenylated at the upstream poly(A) site (for the secreted form) or spliced (for the membrane-bound form) in a mutually exclusive manner. When the mouse mu gene linked to the SV40/HSV-TK hybrid promoter was microinjected into Xenopus oocytes, the mu messenger RNA (mRNA) was altered by coinjection of nuclei of mouse surface IgM-bearing B-lymphoma cells to include the synthesis of the membrane-bound form. An increase in the membrane-bound form was not observed when nuclei of IgM-secreting hybridoma cells or fibroblast cells were coinjected. Deletion of the upstream poly(A) site did not eliminate the effect of B-lymphoma nuclei suggesting that membrane-specific splicing is stimulated. Further, splicing of other mu gene introns was not affected by coinjection of B-lymphoma nuclei. These results suggest that mature B cells contain one or more transacting nuclear factors that stimulate splicing specific for membrane-bound mu mRNA.     

Xenopus oocytes injected with rat uterine RNA express very slowly activating potassium currents

Under the influence of estrogen, uterine smooth muscle becomes highly excitable, generating spontaneous and prolonged bursts of action potentials. In a study of the mechanisms by which this transition in excitability occurs, polyadenylated RNA from the uteri of estrogen-treated rats was injected into Xenopus oocytes. The injected oocytes expressed a novel voltage-dependent potassium current. This current was not observed in oocytes injected with RNA from several other excitable tissues, including rat brain and uterine smooth muscle from ovariectomized rats not treated with estrogen. The activation of this current on depolarization was exceptionally slow, particularly for depolarizations from relatively negative membrane potentials. Such a slowly activating channel may play an important role in the slow, repetitive bursts of action potentials in the myometrium.     

Assembly of functional U1 and U2 human-amphibian hybrid snRNPs in Xenopus laevis oocytes

Oligonucleotides complementary to regions of U1 and U2 small nuclear RNAs (snRNAs), when injected into Xenopus laevis oocytes, rapidly induced the specific degradation of U1 and U2 snRNAs, respectively, and then themselves were degraded. After such treatment, splicing of simian virus 40 (SV40) late pre-mRNA transcribed from microinjected viral DNA was blocked in oocytes. If before introduction of SV40 DNA into oocytes HeLa cell U1 or U2 snRNAs were injected and allowed to assemble into small nuclear ribonucleoprotein particle (snRNP)-like complexes, SV40 late RNA was as efficiently spliced as in oocytes that did not receive U1 or U2 oligonucleotides. This demonstrates that oocytes can form fully functional hybrid U1 and U2 snRNPs consisting of human snRNA and amphibian proteins.     

Differential development of two classes of acetylcholine receptors in Xenopus muscle in culture

Physiological properties of acetylcholine receptors on muscle cells at very early stages of ontogeny were compared with those of cells at later stages. Two changes were observed that contributed to an overall shortening of the mean open time of single-channels. First, there was a shift in the relative proportions of two receptor types with different conductances and mean open times, such that the contribution of receptors with large conductance and short open time increased as development proceeded. Second, there was a sharp reduction in the mean open time of channels having small conductance, with no similar change in channels having large conductance.     

Induction by NGF of meiotic maturation of Xenopus oocytes expressing the trk proto-oncogene product.

The effect of nerve growth factor (NGF) was assessed in Xenopus oocytes expressing the human trk proto-oncogene product, p140prototrk. Oocytes injected with trk messenger RNA expressed polypeptides recognized by antibodies to the trk gene product. Exposure of these oocytes to nanomolar amounts of NGF resulted in specific surface binding of 125I-labeled NGF, tyrosine phosphorylation of p140prototrk, and meiotic maturation, as determined by germinal vesicle breakdown and maturation promoting factor (p34cdc2) kinase activation. Thus the trk proto-oncogene product can act as a receptor for NGF in a functionally productive manner.     

3种性类固醇激素及其受体在北方山溪鲵卵母细胞发育中的分布

为探讨性类固醇激素对北方山溪鲵(B a trachup erus tibetanus)卵泡发育的调控作用,采用组织学与免疫细胞化学方法,观测卵子发生周期中雌二醇(17-βestrad io l,E2)、孕酮(progesterone,P)、睾酮(testosterone,T)3种性类固醇激素及其受体(雌激素受体(E strogen R eceptor,ER)、孕激素受体(P rogesterone R eceptor,PR)、雄激素受体(A ndrogen R eceptor,AR))在不同发育时期卵泡中的表达特征。结果可见,北方山溪鲵卵巢中Ⅰ期卵泡数量在10月达到最大值,Ⅱ期卵泡数量在翌年5月达到最大值,Ⅲ期卵泡数量在6月最多,Ⅳ期卵泡数量在11月最多。E2与ER在Ⅲ期卵泡卵母细胞胞质中的阳性反应最强,P与PR,T与AR均在Ⅳ期卵泡卵母细胞中的阳性反应最强。结果表明,北方山溪鲵的卵泡为异步发育,其从发生至成熟后排出需要的最短时间应为20~22个月。E2在卵母细胞的卵黄形成中起作用,P与T均对卵母细胞成熟起重要作用。3种性类固醇激素分泌的变化规律与北方山溪鲵卵泡发生、发育、成熟和排出的周期一致。     

Cholecystokinin receptors in the brain: characterization and distribution

Specific cholecystokinin binding sites in particulate fractions of rat brain were measured with iodine 125-labeled Bolton-Hunter cholecystokinin, a cholecystokinin analog that has full biological activity. Binding was detected in brain regions known to contain immunoreactive cholecystokinin. Binding was saturable, reversible, of high affinity (dissociation constant, 1.7 x 10(-9) M), and was inhibited by cholecystokinin analogs but not by unrelated hormones.     

Imaging dopamine receptors in the human brain by positron tomography

Neurotransmitter receptors may be involved in a number of neuropsychiatric disease states. The ligand 3-N-[11C]methylspiperone, which preferentially binds to dopamine receptors in vivo, was used to image the receptors by positron emission tomography scanning in baboons and in humans. This technique holds promise for noninvasive clinical studies of dopamine receptors in humans.     

Functional properties of rat brain sodium channels expressed in a somatic cell line

Transfection of Chinese hamster ovary cells with complementary DNA encoding the RIIA sodium channel alpha subunit from rat brain led to expression of functional sodium channels with the rapid, voltage-dependent activation and inactivation characteristic of sodium channels in brain neurons. The sodium currents mediated by these transfected channels were inhibited by tetrodotoxin, persistently activated by veratridine, and prolonged by Leiurus alpha-scorpion toxin, indicating that neurotoxin receptor sites 1 through 3 were present in functional form. The RIIA sodium channel alpha subunit cDNA alone is sufficient for stable expression of functional sodium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

The coupling of neurotransmitter receptors to ion channels in the brain

Recent studies on the action of neurotransmitters on hippocampal pyramidal cells indicate that different neurotransmitter receptors that use either the same or different coupling mechanisms converge onto the same ion channel. Conversely, virtually all of the neurotransmitters act on at least two distinct receptor subtypes coupled to different ion channels on the same cell. The existence of both convergence and divergence in the action of neurotransmitters results in a remarkable diversity in neuronal signaling.     

Solubilized adenosine receptors in the brain: regulation of guanine nucleotides

Adenosine receptors associated with a reduction of adenylate cyclase and labeled by tritium-labeled cyclohexyladenosine can be solubilized from brain membranes with sodium cholate. Regulation of receptor binding by guanine nucleotides is retained in the soluble state. Influences of cations observed in membrane preparations of adenosine receptors are no longer detected with the solubilized receptors. The apparent retention of a complex of receptors and guanosine triphosphate binding but not cation binding protein in the soluble state may permit a molecular analysis of receptor regulation.