Endogenous d-Serine in Rat Brain: N-Methyl-d-Aspartate Receptor-Related Distribution and Aging

Abstract: Recently, a substantial amount of free d -serine has been demonstrated in rat brain, although it has long been presumed that d -amino acids are uncommon in mammals. The anatomical distribution and age-related changes in endogenous d -serine have been examined here to obtain insight into its physiological functions. Free d -serine exclusively occurs in brains, with a persistent high content from birth to at least 86 postnatal weeks. The patterns of the regional variations and the postnatal changes in brain d -serine are closely correlated with those of the N -methyl- d -aspartate (NMDA)-type excitatory amino acid receptor. Because d -serine potentiates NMDA receptor-mediated transmission by selective stimulation of the strychnine-insensitive glycine site of the NMDA receptor, it is proposed that d -serine is a novel candidate as an intrinsic ligand for the glycine site in mammalian brain.     

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D-2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D-2 subunit-specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro-basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit-containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.     

Cloning, Functional Coexpression, and Pharmacological Characterisation of Human cDNAs Encoding NMDA Receptor NR1 and NR2A Subunits

Abstract: Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of rat N -methyl- d -aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C-terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N-terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C-terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors in Xenopus oocytes. These receptors had EC₅₀ values of 2.14 and 2.05 μ M for glutamate and glycine, respectively, and an IC₅₀ of 46.8 μ M for Mg²⁺. Responses were antagonised by d -2-amino-5-phosphonovalerate, L-689,560, pH 6.3, zinc, and MK-801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.     

NMDA受体亚单位NR2B的结构、功能特性及其表达与调控

NMDA受体是兴奋性氨基酸谷氨酸(Glu)的特异性受体,属配体门控离子通道,是由不同的亚单位组成.现已发现,NMDA受体至少存在7个亚单位(NR1,NR2A-D,NR3A-B),其中NR2B在7个亚单位中扮演非常重要的角色.近年来对NR2B研究表明,其在调控神经元突触的可塑性、学习与记忆以及治疗精神紊乱方面具有重要的意义.对近期有关NR2B亚单位的结构、功能特性及其表达与调控的研究进展做一综述.     

Molecular Cloning and Regional Distribution of a Human Proton Receptor Subunit with Biphasic Functional Properties

Abstract : Small changes of extracellular pH activate depolarizing inward currents in most nociceptive neurons. It has been recently proposed that acid sensitivity of sensory as well as central neurons is mediated by a family of proton-gated cation channels structurally related to Caenorhabditis elegans degenerins and mammalian epithelial sodium channels. We describe here the molecular cloning of a novel human proton receptor, hASIC3, a 531-amino acid-long subunit homologous to rat DRASIC. Expression of homomeric hASIC3 channels in Xenopus oocytes generated biphasic inward currents elicited at pH <5, providing the first functional evidence of a human proton-gated ion channel. Contrary to the DRASIC current phenotype, the fast desensitizing early component and the slow sustained late component differed both by their cationic selectivity and by their response to the antagonist amiloride, but not by their pH sensitivity (pH₅₀ = 3.66 vs. 3.82). Using RT-PCR and mRNA blot hybridization, we detected hASIC3 mRNA in sensory ganglia, brain, and many internal tissues including lung and testis, so hASIC3 gene expression was not restricted to peripheral sensory neurons. These functional and anatomical data strongly suggest that hASIC3 plays a major role in persistent proton-induced currents occurring in physiological and pathological conditions of pH changes, likely through a tissue-specific heteropolymerization with other members of the proton-gated channel family.     

The Mouse GalR2 Galanin Receptor: Genomic Organization, cDNA Cloning, and Functional Characterization

Abstract: The diverse physiological actions of galanin are thought to be mediated through activation of galanin receptors (GalRs). We report the genomic and cDNA cloning of a mouse GalR that possesses a genomic structure distinct from that of GalR1 and encodes a functional galanin receptor. The mouse GalR gene consists of two exons separated by a single intron within the protein-coding region. The splicing site for the intron is located at the junction between the third transmembrane domain and the second intracellular loop. The cDNA encodes a 370-amino acid putative G protein-coupled receptor that is markedly different from human GalR1 and rat GalR3 (38 and 57%) but shares high homology with rat GalR2 (94%). In binding studies utilizing membranes from COS-7 cells transfected with mouse GalR2 cDNA, the receptor displayed high affinity ( K _D = 0.47 n M ) and saturable binding with ¹²⁵I-galanin ( B _max = 670 fmol/mg). The radioligand binding can be displaced by galanin and its analogues in a rank order: galanin ⋍ M40 ⋍ M15 ⋍ M35 ⋍ C7 ⋍ galanin (2–29) ⋍ galanin (1–16) ≫ galanin (10–29) ⋍ galanin (3–29), which resembles the pharmacological profile of the rat GalR2. Receptor activation by galanin in COS-7 cells stimulated phosphoinositide metabolism, which was not reversed by pertussis toxin. Thus, the galanin receptor encoded in the cloned mouse GalR gene is the type 2 galanin receptor and is active in both ligand binding and signaling assays.     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

Monoclonal Antibody Analysis of Phosphatidylserine and Protein Kinase C Localizations in Developing Rat Cerebellum

N-Methyl-D-aspartate (NMDA) stimulated the release of endogenous dopamine from striatal slices prepared from adult Sprague-Dawley rats. A mixture of sodium fluoride and aluminum chloride (AlF4-) added to the slices significantly potentiated the NMDA-stimulated release of dopamine in a concentration- and time-dependent manner. The AlF4- mixture had no effect on the nonstimulated basal efflux of dopamine, and no increases in NMDA-stimulated release were observed when NaF was replaced with NaCl. Similarly, AlCl3 or a mixture of NaCl and AlCl3 had no effect on NMDA-stimulated release. The AlF(4-)-induced increase in NMDA-stimulated dopamine release was totally blocked by magnesium or the selective NMDA glycine antagonist 7-chlorokynurenic acid. Striatal slices depolarized with KCl (15 mM) also released dopamine and this release was similarly potentiated by AlF4-. However, KCl-stimulated dopamine release from striatal synaptosomes was not potentiated by concentrations of AlF4- that greatly increased release from striatal slices. NMDA did not stimulate the release of dopamine from striatal synaptosomes in the absence or presence of aluminum fluoride. Modulators of adenylate cyclase (forskolin) and protein kinase C (phorbol esters) did not enhance NMDA-stimulated dopamine release. The protein kinase C inhibitor H-7 also did not reduce the potentiating effects of AlF4-. The mixed cholinergic agonist carbachol and the calcium ionophore A23187 mimicked the AlF4- effect although the increase in NMDA-stimulated dopamine release produced by these agents was less than that seen with AlF4-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression and Functional Analysis of Toll-like Receptor 4 in Human Cervical Carcinoma

Toll-like receptors are expressed in human immune cells and many tumors, but the role of toll-like receptor 4 (TLR4) in the development of tumors is controversial. We demonstrated the expression, distribution, and functional activity of TLR4 in tissues of normal cervix, cervical intraepithelial neoplasia (CIN), invasion cervical cancers (ICC), and different human papillomavirus (HPV)-infected cervical cancer cells. The results showed that TLR4 expression was in accordance with the histopathological grade: higher in ICC than in CIN, and low in normal cervical tissues and malignant cervical stroma. Expression was higher in SiHa (HPV16+) than in HeLa (HPV18+) cells, but was not observed in C33A (HPV?) cells. After treatment with its agonist, lipopolysaccharide (LPS), the expression levels of TLR4 was increased and apoptosis resistance was induced in SiHa cells, but not in HeLa or C33A cells. Meanwhile, LPS treatment did not alter the cell cycle distribution in SiHa cells. The mechanism of apoptosis resistance may be related to HPV16 infection and not correlated with the cell cycle distribution. Targeting TLR4 in combination with traditional drug treatment may serve as a novel strategy for more effectively killing cancer cells.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

The Expression of Two Splice Variants of Metabotropic Glutamate Receptor Subtype 5 in the Rat Brain and Neuronal Cells During Development

Abstract: We previously reported that a variant with extra amino acid residues exists in the metabotropic glutamate receptor subtype 5 (mGluR5). Either of the two isoforms, named mGluR5b and mGluR5a for the isoforms with and without the inserted sequence, respectively, generated Ca²⁺-activated Cl⁻ current when expressed in Xenopus oocytes. We herein report that these two isoforms are produced by the alternative splicing of the exon skipping type. When examined during the course of postnatal development, the major mGluR5 isotype mRNA was observed to switch from mGluR5a to mGluR5b in the rat hippocampus and the cerebral cortex. We also investigated two cell lines that could be differentiated into neuron-like cells in vitro. Whereas the mGluR5b mRNA was hardly detectable in either undifferentiated or differentiated NG108-15 cells, the relative amounts of the two variant mRNAs changed after the induction of differentiation in the P19 cells. An extracellular application of trans - d,l -1-amino-1,3-cyclopentanedicarboxylate on the neuron-like P19 cells induced intracellular Ca²⁺ mobilization, thus suggesting that the cells could express functional mGluR(s) coupled to phospholipase C and other components that could mediate the signal transduction pathway. This cell line may thus provide a model system for studying both mGluR5 expression and other mGluR-induced phenomena at the molecular level.     

Distribution of the Glucose-1,6-Bisphosphate System in Brain and Retina

The distribution of glucose-1,6-bisphosphate (G16P2) synthase was measured in more than 70 regions of mouse brain, and nine layers of monkey retina. Activities in gray areas varied as much as 10-fold, in a hierarchical manner, from highest in telencephalon, especially the limbic system, to lowest in cerebellum, medulla, and spinal cord. The synthase levels were significantly correlated among different regions with G16P2 itself, as well as with previously published levels of a brain specific IMP-dependent G16P2 phosphatase. In contrast, neither G16P2 nor either its synthase or phosphatase correlated positively with phosphoglucomutase, and in all regions the G16P2 levels greatly exceeded requirements for activation of this mutase. This strengthens the view that G16P2 has some function besides serving as coenzyme for phosphoglucomutase. However, attempts to correlate the "G16P2 system," as defined by the three coordinately related elements, synthase, phosphatase, and G16P2, with other enzymes of carbohydrate metabolism, or with regional data of Sokoloff et al. [J. Neurochem. 28, 897-916 (1977)] for glucose consumption, were unsuccessful. This leaves open the possibility that brain G16P2 might serve as a phosphate donor for specific nonmetabolic effector proteins.     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

Transglutaminase Activity in Rat Brain: Characterization, Distribution, and Changes with Age

The activity of transglutaminase was characterized in the rat brain. In adults, comparable levels of transglutaminase activity are present in all brain regions examined. The activity is present in all subcellular fractions, as studied by differential centrifugation, but the soluble fraction contains the highest specific activity. The endogenous activity (enzyme activity assayed in the absence of the exogenous substrate casein) is very low in all subcellular fractions, except in the synaptosomal fraction where its highest levels are about 40-60% of the activity assayed in the presence of casein. Furthermore, enzyme activity is present on the external surface of synaptosomes. In the soluble fraction, maximal activity can be detected between pH values of 9 and 10 when assayed in the presence of 5 mM CaCl2 (with half-maximal activity requiring 0.75 mM CaCl2) and 0.4 mM putrescine (with an apparent Km for putrescine of 0.1 mM). The activity can be partially inhibited by ZnCl2 (with an IC50 of 4.5 mM) and by AlCl3 (with an IC50 of 5.1 mM). In the cerebellum, where the full span of neuronal development can be studied after birth, the highest specific activity is observed just after birth, thereafter the activity starts to decline and by 14 days, after a reduction of about 65%, it reaches levels observed throughout life.     

Regional and Ontogenic Expression of the NMDA Receptor Subunit NR2D Protein in Rat Brain Using a Subunit-Specific Antibody

Abstract: A polyclonal antibody for the NMDA receptor subunit NR2D has been developed that identifies an ∼160-kDa band on immunoblots from NR2D transfected cells and CNS tissues. No cross-reactivity is seen with other NMDA receptor subunits. The NR2D receptor subunit is N -glycosylated in both brain and transfected cells. Transfected cells expressing NR2D are immunofluorescently labeled, whereas untransfected cells or cells transfected with other NMDA receptor subunit cDNAs are not. Similarly, the NR2D subunit is selectively and quantitatively immunoprecipitated, whereas the NR1, NR2A, or NR2B subunit is not. The relative densities of the NR2D subunit in nine areas of postnatal day 7 and adult rat brains have been determined by quantitative immunoblotting. NR2D was expressed at highest levels in the thalamus, midbrain, medulla, and spinal cord, whereas intermediate levels of this subunit were found in the cortex and hippocampus. Low or undetectable levels were seen in the olfactory bulb, striatum, and cerebellum. Following a peak after the first week of birth, NR2D protein levels decreased by about twofold in adulthood in all rat brain regions examined. More complete ontogenic profiles were determined for the diencephalon, telencephalon, and spinal cord where similar ontogenic patterns were seen. NR2D protein is present at high levels at embryonic stages of development, rises to a peak at postnatal day 7, and decreases but remains measurable during late postnatal life. This study demonstrates the generation and characterization of an antibody selective for the NR2D NMDA receptor subunit as well as a determination of the distribution and ontogenic profile of this subunit in rat brain. The results suggest that native NMDA receptors containing the NR2D subunit may have functional roles not only in the young brain but also in adult brain.     

Regional Distribution and Production Rate of 3-Methoxy-4-Hydroxyphenylethyleneglycol Sulphate (MHPG-SO4) in Rat Brain

The levels of noradrenaline (NA) and 3-methoxy-4-hydroxyphen-ylethyleneglycol sulphate (MHPG-SO₄) in 15 brain regions showed a parallel distribution in male Wistar rats. The differences in regional distribution of MHPG-SO₄ were similar to those in the rate of NA turnover reported by other investigators. The accumulation rates of MHPG-SO₄ during 45 and 90 min after probenecid injection significantly correlated to the steady state levels of MHPG-SO₄ in nine regions studied. With the results, the regional levels of MHPG-SO, either in untreated or in probenecid-treated rats, are considered to be a useful index of NA turnover.     

p53、BCL-2、Caspase－3等凋亡相关蛋白在大鼠脑损伤中的表达及意义

目的：检测脑外伤大鼠中p53、bcl-2及caspase-3表达,并分析其与脑外伤之间的关系,为脑损伤患者预后提供部分参数依据。方法：建立脑外伤大鼠实验动物模型,用免疫组化方法检测p53、Bcl-2和Caspase-3的表达。结果：脑创伤后在创伤周围区的神经元会发生凋亡,凋亡的发生可能与p53、bcl-2、caspase-3等基因及蛋白的调节有关。结论：脑创伤后在创伤周围区的神经元会发生凋亡,凋亡的发生可能与p53、bcl-2、caspase-3等基因及蛋白的调节有关。     

人结合珠蛋白cDNA克隆及其在大肠杆菌中的表达和鉴定

目的：克隆人结合珠蛋白（haptoglobin,Hp）cDNA,并在大肠杆菌中表达和鉴定。方法：从Hela细胞中分离总RNA,采用RT-PCR方法获得人Hp cDNA,分别克隆至原核表达载体pET-32a和PGEX-4T-1,转化至大肠杆菌BL21,IPTG诱导表达,并进行SDS—PAGE及Western blot鉴定。结果：成功构建了高效原核表达质粒PET-32a-Hp和PGEX-4T1-Hp;Western印迹结果表明,经IPTG诱导,在大肠杆菌中表达了分子量约30kD和37kD的目的蛋白;表达产物经Ni2^＋-NTA离子交换树脂纯化,纯度〉90％。结论：在E．coli成功表达和纯化了人Hp融合蛋白,为进一步开发人Hp诊断试剂打下基础。     

The Pyruvate Dehydrogenase Complex: Cloning of the Rat Somatic El α Subunit and Its Coordinate Expression with the mRNAs for the E1β E2, and E3 Catalytic Subunits in Developing Rat Brain

Abstract: We report the isolation of cDNA clones encoding the somatic form of the E1α subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1α sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1α subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1α cDNA, in conjunction with cDNA probes to the E1β, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six-and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.