Expression of the Raf-1 protein in rat brain during development and its hormonal regulation in hypothalamus.

To study mechanisms involved in the sexual differentiation of the rat brain, the expression of the protein product of the proto-oncogene c-raf-1 (Raf-1) was examined. Biochemical and immunocytochemical analyses localized Raf-1 in embryonic rat brain regions and demonstrated hormonally induced changes in Raf-1 expression. For this study an affinity-purified anti-peptide antiserum specific for Raf-1 (NH-44) was used. Western blots revealed an approximately 77 kD polypeptide isolated in the cytosol of developing rat brains. Raf-1 levels were highest in the embryonic (E) day 22 female hypothalamus (HYP), and approximately twofold higher than levels detected in male HYP at E22 as determined by quantitative protein dot blot and semiquantitative Western blot analyses. Raf-1 levels in HYP were greater than those in either brain stem (BS) or cortex. Immunocytochemical analysis revealed high levels of Raf-1 in selective brain regions (e.g., the ventromedial nucleus in the HYP, the mitral cell layers in the main and accessory olfactory bulbs (OB), and the locus coeruleus) at E22 and postnatal (P) day 1. Lower levels of immunoreactivity were observed in many areas of the perinatal neuraxis. To test hormonal regulation of Raf-1, testosterone propionate (TP) was administered to pregnant rats on E17; male and female fetuses were examined on E22. This treatment significantly decreased Raf-1 levels in female HYP, but not in male HYP, as determined by Western blot analysis. No significant sex difference or response to prenatal hormone treatments were observed in either brain stem or cortex. No significant sex difference was noted postnatally, and administration of TP 3 h after birth did not change Raf-1 levels examined 24 h later. In summary, Raf-1 was localized within selective regions of the rat brain, and its expression was altered by exogenous prenatal hormonal stimulation. One role for Raf-1 in signal transduction may be to delimit hormonal critical periods in sexual differentiation of the brain.     

Cellular distribution of NDRG1 protein in the rat kidney and brain during normal postnatal development.

N-myc downregulated gene 1 (NDRG1) is a 43-kD protein whose mRNA is induced by DNA damage, hypoxia, or prolonged elevation of intracellular calcium. Although NDRG1 is also upregulated during cell differentiation, there are few studies on NDRG1 expression during postnatal development. Here we investigated the expression and cellular distribution of NDRG1 protein in rat kidney and brain during postnatal development. Immunohistochemical analysis revealed that the cellular localization of NDRG1 protein in the kidney changed from the proximal convoluted tubules to the collecting ducts between postnatal days 10 and 20. In the brain, a change in cellular expression was also found from the hippocampal pyramidal neurons to the astrocytes in the gray matter during the same postnatal period. These alterations in the cellular distribution of NDRG1 were associated with shifts in the molecular assembly on Western blots. Under non-reduced conditions, the main NDRG1 band was found only around 215 kD in both kidney and brain during the early postnatal stage. After postnatal day 10, the immunoreactive bands shifted to 43 kD in the kidney and 129 kD in the brain. These changes in the cellular distribution and state of assembly may correlate with the functional maturation of both organs.     

Expression pattern of ACK1 tyrosine kinase during brain development in the mouse

Ack1 is a non-receptor tyrosine kinase that is highly expressed in the adult central nervous system (CNS). Here, we studied the distribution of Ack1 mRNA throughout the development of mouse CNS. Expression was detected in all areas of the brain but especially high levels were observed in the neocortex, hippocampus, and cerebellum. Interestingly, expression levels were prominent in areas of proliferation such as the subventricular zone and areas that originate other structures such the pontine nucleus and the ganglionic eminence. During development, several areas showed an increase in Ack1 expression, especially the dentate gyrus and CA3 in the hippocampus, layer V in the neocortex, and the Purkinje cell layer in the cerebellum. These results demonstrate that this kinase is up-regulated during development and that it is expressed in proliferative areas and in migratory pathways in the developing brain.     

Hexose diphosphates and phosphofructokinase in rat brain during development

Fructose 2,6-diphosphate and glucose 1,6-diphosphate concentrations were determined during late gestation and over the course of suckling in rat brain cortex and cerebellum. Cortex fructose 2,6-diphosphate concentration was greatest in neonatal animals and gradually declined thereafter by 25% to reach the adult level at 15 days of age. In contrast, the glucose 1,6-diphosphate concentration increased 4-fold over the same period to reach its highest level by postnatal day 15. Neither cerebellar fructose 2,6-diphosphate nor glucose 1,6-diphosphate concentrations varied significantly. Six day cortex 6-phosphofructo-1-kinase was less sensitive to inhibition by citrate than the enzyme obtained from 15 day pups, and fructose 2,6-diphosphate was better than glucose 1,6-diphosphate at relieving the inhibition imposed by citrate at either age. It is suggested that the rise in cerebral glucose use which occurs during suckling cannot be attributed to either changes in the concentrations of fructose 2,6-diphosphate or glucose 1,6-diphosphate, or the age-related differential sensitivity of 6-phosphofructo-1-kinase toward these effectors.     

大鼠脑内caveolin-1蛋白的表达及其在分辨学习中的作用

Caveolin-1（Cav—1）蛋白作为细胞质膜结构小窝（caveolae）的标志蛋白,在胆固醇运输、膜组装、信号转导和细胞转化过程中扮演重要的角色。为了探讨Cav-1蛋白在中枢神经系统可塑性及学习记忆中的作用,本文以Sprague—Dawley大鼠为实验对象,利用蛋白质免疫印迹杂交方法观察了Car-1蛋白在不同年龄大鼠脑内表达的特征,并研究了Y-迷宫训练前后Cav-1蛋白表达的变化。结果表明：（1）大鼠不同脑区Cav-1蛋白表达的年龄特征不同。海马内的表达属青年鼠最高,其次是老年鼠和幼年鼠;皮层内的表达属幼年鼠最高,其次是老年鼠,青年鼠最低;小脑内的表达无明显年龄差异。（2）Y-迷宫训练引起青年鼠海马和前额叶皮层内Cav-1蛋白的表达显著增加。结果提示,Cav-1蛋白与动物脑发育和学习记忆有密切关系,可能参与中枢可塑性的调节。     

Characterization of a novel putative zinc finger gene MIF1: involvement in multiple hormonal regulation of Arabidopsis development

Phytohormones play crucial roles in regulating many aspects of plant development. Although much has been learned about the effects of individual hormones, cross-talk between and integration of different hormonal signals are still not well understood. We present a study of MINI ZINC FINGER 1 (MIF1), a putative zinc finger protein from Arabidopsis, and suggest that it may be involved in integrating signals from multiple hormones. MIF1 homologs are highly conserved among seed plants, each characterized by a very short sequence containing a central putative zinc finger domain. Constitutive overexpression of MIF1 caused dramatic developmental defects, including dwarfism, reduced apical dominance, extreme longevity, dark-green leaves, altered flower morphology, poor fertility, reduced hypocotyl length, spoon-like cotyledons, reduced root growth, and ectopic root hairs on hypocotyls and cotyledons. In addition, 35S::MIF1 seedlings underwent constitutive photomorphogenesis in the dark, with root growth similar to that in the light. Furthermore, 35S::MIF1 seedlings were demonstrated to be non-responsive to gibberellin (GA) for cell elongation, hypersensitive to the GA synthesis inhibitor paclobutrazol (PAC) and abscisic acid (ABA), and hyposensitive to auxin, brassinosteroid and cytokinin, but normally responsive to ethylene. The de-etiolation defect could not be rescued by the hormones tested. Consistent with these observations, genome-scale expression profiling revealed that 35S::MIF1 seedlings exhibited decreased expression of genes involved in GA, auxin and brassinosteroid signaling as well as cell elongation/expansion, and increased expression of ABA-responsive genes. We propose that MIF1, or the protein(s) with which MIF1 interacts, is involved in mediating the control of plant development by multiple hormones.     

Growth regulation and development in Physcomitrella patens: an insight into growth regulation and development of bryophytes

Observations made on the effects of auxin, of cytokinin and of various light treatments on cultures of wild-type Physcomitrella patens , grown either on solid medium or in continuously-replaced liquid medium, have allowed us to suggest probable roles for these factors during the early stages of normal gametophyte development. Further data obtained by studying mutants affected in their responses to one or both types of hormones or to light have supported our model of gametophyte development and enabled us to elaborate it.     

The relative proportion of H1° and A24 is reversed in oligodendrocytes during rat brain development

1. The histone complement of oligodendrocyte chromatin at different stages of brain development was studied after acid extraction of nuclei. 2. HCl-soluble proteins were analyzed by different electrophoretic techniques. 3. Our results show an increase in the concentration of histone H1(0) with differentiation. 4. The increase in H1(0) is accompanied by a concomitant decrease in the total amount of the ubiquitinated form of histone H2A (A24).     

Expression of Msx1 and Dlx1 during Dumbo rat head development: Correlation with morphological features

The Dumbo rat possesses some characteristics that evoke several human syndromes, such as Treacher-Collins: shortness of the maxillary, zygomatic and mandibular bones, and low position of the ears. Knowing that many homeobox genes are candidates in craniofacial development, we investigated the involvement of the Msx1 and Dlx1 genes in the Dumbo phenotype with the aim of understanding their possible role in abnormal craniofacial morphogenesis and examining the possibility of using Dumbo rat as an experimental model for understanding abnormal craniofacial development. We studied the expression of these genes during craniofacial morphogenesis by RT-PCR method. We used Dumbo embryos at E12 and E14 and included the Wistar strain as a control. Semi-quantitative PCR analysis demonstrated that Msx1 and Dlx1 are expressed differently between Dumbo and Wistar rats, indicating that their low expression may underly the Dumbo phenotype.     

Telencephalic and diencephalic origin of radial glial processes in the developing preoptic area/anterior hypothalamus

Neuronal birth-dating sudies using [³H]thymidine have indicated that neurons in the preoptic area/anterior hypothalamus (POA/AH) are derived primarily from progenitors in proliferative zones surrounding the third ventricle. Radial glial processes are potential guides for neuronal migration, and their presence and orientation during development may provide further information about the origin of cells in the POA/AH. In addition to determining the orientation of radial glial fibers, we examined the relationship of neurons with identified birth dates to radial glial processes in the developing POA/AH of ferrets. Neuronal birth dates were determined by injecting ferret fetuses with bromodeoxyuridine (BrdU) at several different gestational ages; brains were taken from ferret kits at subsequent prenatal ages. Sections were processed for immunocytochemistry to reveal vimentin or glial fibrillary acidic protein in radial glia, or BrdU-labeled cell nuclei. Numerous radial glial processes extended from the lateral ventricles through ventral portions of the septal region to the pial surface of the POA/AH. These fibers both encapsulated and coursed ventrally through and around the anterior commissure of ferret, rat, and mouse fetuses. These ventrally directed fibers were less evident at older ages. In double-labeled sections from ferrets, BrdU-labeled cells in the dorsal POA/AH were often aligned in the same dorsal-ventral orientation as adjacent radial glial fibers. We suggest that a subset of neurons, originating in telencephalic proliferative zones, migrates ventrally along radial glial guides into the dorsal POA/AH. © 1995 John Wiley & Sons, Inc.     

A gene trap knockout of the Tiam-1 protein results in malformation of the early embryonic brain

Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice.     

烫伤对大鼠下丘脑室旁核内皮素—1合成和分泌的影响

目的研究烫伤后下丘脑室旁核(PVH)内皮素-1(ET-1)的合成和分泌改变,探讨PVHET-1在烫伤中的病理生理学意义。方法用原位杂交和免疫组织化学方法观察了烫伤后PVHET-1合成和分泌的变化,并用通用图象颗粒分析法检测单位面积内ET-1mRNA阳性杂交信号的强度和ET-1样免疫反应物(ET-1-ir)免疫反应强度。结果烫伤后15minPVH神经元胞浆内ET-1mRNA阳性杂交信号与对照组相比未见明显差异,烫伤后60min和180minPVH神经元胞浆内ET-1mRNA阳性杂交信号较对照组(100％±25％)明显增多,强度明显增高,分别为138％±26％(P<0．05)和167％±18％(P<0.01);而烫伤后15minPVH神经元胞浆内ET-1阳性反应物明显减少,免疫反应物强度为6．3％±1．5％,显著低于对照组(P<0．01),烫伤后60min和180min逐渐回升,分别为23．1％±2．9％和44．1％±3．8％,但仍显著低于对照组(P＜0．01)。结论烫伤后PVHET-1合成和分泌增加。     

Expression of estrogen receptor-alpha and -beta mRNA in the brain of Japanese quail embryos

The present study was conducted to investigate the mRNA expression of the two estrogen receptor (ER) subtypes ERalpha and ERbeta in the brain of Japanese quail embryos. We found expression of both ERalpha and ERbeta mRNA in homogenate of whole head from 6-day-old embryos, and in brain homogenate from 9- and 12-day-old embryos using real-time PCR. In 9- and 12-day-old embryos the ERalpha expression was higher in females than in males. We used in situ hybridization to examine the localization of the ERs in sections from male and female brains on day 9 and day 17 of incubation. On day 9, ERbeta mRNA was detected in the developing medial preoptic nucleus (POM), in the medial part of the bed nucleus of the striae terminalis (BSTm), and in the tuberal region of the hypothalamus. ERalpha signal could not be detected in the POM, the BSTm or the tuberal region in 9-day-old embryos. In 17-day-old embryos, ERbeta was highly expressed in the preoptic area, the nucleus Taeniae of the Amygdala (TnA) and the BSTm. Expression of ERalpha mRNA was detected in parts of the preoptic area and in the telencephalic TnA. No ERalpha expression was found in the BSTm, an area known to be sexually dimorphic in adults. The high embryonic expression of ERbeta in brain areas linked to sexual behavior indicates that ERbeta plays a role in sexual differentiation of the Japanese quail brain.     

基质Gla蛋白在大鼠附睾发育过程中的表达及定位

目的明确基质Gla蛋白(matrix Gla protein,MGP)在大鼠附睾发育过程中的表达特征。方法采用实时定量PCR和免疫荧光染色方法,对MGP在大鼠附睾不同发育阶段的表达及定位进行检测。结果实时定量PCR结果显示,MGP mRNA在6d、10d、3w、5w、7w、8w、10w和12w的大鼠附睾中均有表达,其表达量在3w达到最高峰,3w至8w表达量逐渐降低,成年大鼠(10～12w)MGP的表达量逐渐升高并稳定在较高水平。免疫荧光染色显示MGP在10d、3w的大鼠附睾各个节段均有表达,在7w、12w的表达主要集中于大鼠附睾体部和尾部,且MGP定位于附睾上皮主细胞和亮细胞。结论MGP在大鼠附睾发育的关键分化期高表达,成年后主要定位于附睾体部和尾部的主、亮细胞,可能对附睾的形态发育和管腔钙稳态的维持起重要作用。     

Expression of second messenger- and cyclin- dependent protein kinases during postnatal development of rat heart

During early postnatal development, cardiomyocytes, which comprise about 80% of ventricular mass and volume, become phenotypically developed to facilitate their contractile functions and terminally differentiated to grow only in size but not in cell number. These changes are due to the expression of contractile proteins as well as the regulation of intracellular signal transduction proteins. In this study, the expression patterns of several protein kinases involved in various cardiac functions and cell-cycle control were analyzed by Western blotting of ventricular extracts from 1-, 10-, 20-, 50-, and 365-day-old rats. The expression level of cAMP-dependent protein kinase was slightly decreased (20%) over the first year, whereas no change was detected in cGMP-dependent protein kinase I. Calmodulin-dependent protein kinase II, which is involved in Ca²⁺ uptake into the sarcoplasmic reticulum, was increased as much as ten-fold. To the contrary, the expressions of protein kinase C-α and ι declined 77% with age. Cyclin-dependent protein kinases (CDKs) such as CDK1, CDK2, CDK4, and CDK5, which are required for cell-cycle progression, abruptly declined to almost undetectable levels after 10–20 days of age. In contrast, other CDK-related kinases, such as CDK8 or Kkialre, did not change significantly or increased up to 50% with age, respectively. Protein kinases implicated in CDK regulation such as CDK7 and Wee1 were either slightly increased in expression or did not change significantly. All of the proteins that were detected in ventricular extracts were also identified in isolated cardiac myocytes in equivalent amounts and analyzed for their relative expression in ten other adult rat tissues. J. Cell. Biochem. 69:506–521, 1998. © 1998 Wiley-Liss, Inc.     

阿片受体样受体ORL1基因在大鼠脑内的表达

我们采用地主辛标记的寡聚核苷酸探针和原位杂产技术研究了新发现的阿片受体样受体ＯＲＬ１基因在正常大鼠脑仙的表达和分布。发现ＯＲＬ１基因在大鼠脑内广泛表达。尤其在大脑皮层、丘脑、下丘脑、杏仁核、海马、隔核、缰核、导水管周围灰质、中缝核群及蓝斑等区域。提示ＯＲＬ１除参与痛觉调制外,还可能参与脑内多项生理功能的调控。