Molecular cloning of a novel immunoglobulin superfamily gene preferentially expressed by brain and testis

We have cloned and characterized a novel gene from both human and mouse that encodes a new member of the immunoglobulin superfamily. The gene is preferentially expressed in both brain and testis, and hence, termed BT-IgSF (brain- and testis-specific immunoglobulin superfamily). The predicted protein consists of V-type and C2-type immunoglobulin domains as well as a hydrophobic signal sequence, a single transmembrane region, and a cytoplasmic domain. Human BT-IgSF protein (431 amino acids) is 88% identical to the mouse protein (428 amino acids) and both show significant homology to coxsackie and adenovirus receptor (CAR) and endothelial cell-selective adhesion molecule (ESAM). We examined the expression of BT-IgSF with various cultured cells and found that the gene was expressed in both neurons and glial cells in vitro. Furthermore, the expression was preferentially detected in pyramidal cell layers of the dentate gyrus and hippocampus and in commissure fibers of the corpus callosum, in brain tissue sections examined. These findings suggest that BT-IgSF plays a role in the development or function of the central nervous system.     

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit the MEKK1/MEK4/JNK signaling pathway in endotoxin-activated microglia

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides, act as anti-inflammatory factors for activated microglia, by inhibiting the production of proinflammatory factors. In the present study the effects of VIP/PACAP on the MEKK1/MEK4/JNK transduction pathway and on the subsequent changes in Jun family members, a transduction pathway clearly involved in the activation of microglia cells were examined. VIP/PACAP inhibit MEKK1 activity and the subsequent phosphorylations of MEK4, JNK, and c-Jun, which result in a decrease in the AP-1 binding and a marked change in the composition of AP-1 complexes from c-Jun/c-Fos to JunB/c-Fos. Furthermore, VIP stimulates JunB production in LPS-stimulated microglia. Both inhibition of the MEKK1/MEK4/JNK pathway, leading to a reduction in phosphorylated c-Jun, and the stimulation of JunB are mediated through the specific VPAC1 receptor and cAMP/PKA pathway. The VIP/PACAP interference with the stress-induced SAPK/JNK pathway in activated microglia may represent a significant element in the regulation of inflammatory response in the CNS by endogenous neuropeptides.     

Molecular cloning of cDNAs encoding variants of meiotin-1

Meiotin-1 is a chromatin-associated protein, originally isolated from microsporocytes of Lilium longiflorum, which is found predominantly in cells undergoing meiotic prophase. Chromatin fractionation studies demonstrated that meiotin-1 has an unusual stoichiometry relative to that of histone H1 and the core histones in chromatin fibers. The protein is found less frequently than is histone H1, and appears to be distributed once every 5 to 13 nucleosomes. This distribution may approximate the number of nucleosomes per turn of the chromatin solenoid. A truncated cDNA was identified by immunoscreening of an expression library, and the cDNA was used as a hybridization probe to select a full length cDNA. Variations between the sequence of the predicted polypeptide and sequenced peptides, and variations between the amino acid composition of the protein and the deduced protein indicate that the cDNAs encode minor variants of mature meiotin-1. RNA gel blot hybridization studies reveal that the meiotin-1 mRNA is restricted to anthers in which meiosis is occurring. Computer analysis of the polypeptide deduced from the cDNA indicates that the protein begins with a region highly homologous to the conserved central globular domain of histone H1 molecules. DNA gel blotting experiments demonstrate that homologous sequences exist in the genomes of a fern, a fungus, and both mono-and dicotyledonous plants. Meiotin-1 has been evolutionarily conserved and I propose that it arose from histone H1 to fulfill a role in organizing meiotic chromatin.     

Endogenous reactive oxygen species cause astrocyte defects and neuronal dysfunctions in the hippocampus: a new model for aging brain

The etiology of astrocyte dysfunction is not well understood even though neuronal defects have been extensively studied in a variety of neuronal degenerative diseases. Astrocyte defects could be triggered by the oxidative stress that occurs during physiological aging. Here, we provide evidence that intracellular or mitochondrial reactive oxygen species (ROS) at physiological levels can cause hippocampal (neuronal) dysfunctions. Specifically, we demonstrate that astrocyte defects occur in the hippocampal area of middle‐aged Tet‐mev‐1 mice with the SDHC^V69E mutation. These mice are characterized by chronic oxidative stress. Even though both young adult and middle‐aged Tet‐mev‐1 mice overproduced MitoSOX Red‐detectable mitochondrial ROS compared to age‐matched wild‐type C57BL/6J mice, only young adult Tet‐mev‐1 mice upregulated manganese and copper/zinc superoxide dismutase (Mn‐ and Cu/Zn‐SODs) activities to eliminate the MitoSOX Red‐detectable mitochondrial ROS. In contrast, middle‐aged Tet‐mev‐1 mice accumulated both MitoSOX Red‐detectable mitochondrial ROS and CM‐H₂DCFDA‐detectable intracellular ROS. These ROS levels appeared to be in the physiological range as shown by normal thiol and glutathione disulfide/glutathione concentrations in both young adult and middle‐aged Tet‐mev‐1 mice relative to age‐matched wild‐type C57BL/6J mice. Furthermore, only middle‐aged Tet‐mev‐1 mice showed JNK/SAPK activation and Ca²⁺ overload, particularly in astrocytes. This led to decreasing levels of glial fibrillary acidic protein and S100β in the hippocampal area. Significantly, there were no pathological features such as apoptosis, amyloidosis, and lactic acidosis in neurons and astrocytes. Our findings suggest that the age‐dependent physiologically relevant chronic oxidative stress caused astrocyte defects in mice with impaired mitochondrial electron transport chain functionality.     

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific

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IM-only protein

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(FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12–q13 by fluorescent in-situ hybridization (FISH).     

Molecular cloning of novel Monad binding protein containing tetratricopeptide repeat domains

We have previously reported that Monad, a novel WD40 repeat protein, potentiates apoptosis induced by tumor necrosis factor-alpha(TNF-alpha) and cycloheximide (CHX). By affinity purification and mass spectrometry, we identified RNA polymerase II-associated protein 3 (RPAP3) as a binding protein of Monad. Overexpression of RPAP3 in HEK 293 potentiated caspase-3 activation and apoptosis induced by TNF-alpha and CHX. In addition, knockdown of RPAP3 by RNA interference resulted in a significant reduction of apoptosis induced by TNF-alpha and CHX in HEK293 and HeLa cells. These results raise the possibility that RPAP3, together with Monad, may function as a novel modulator of apoptosis pathway.     

The role of protein kinase SAPK/JNK in cell responses to low-intensity nonionizing radiation

The effect of low-intensity laser light (He-Ne, 0.2 mW/cm², 632.8 nm, exposure time 1 min) or centimeter waves (8.15–18 GHz, 1 μW/cm², exposure time 1 h) on Phospho-SAPK/JNK production in mice lymphocytes was investigated. Normal isolated spleen lymphocytes or cells incubated previously with geldanamycin, an inhibitor of Hsp90, were used in the experiments. Significant stimulation of Phospho-SAPK/JNK production in lymphocytes after treatment with laser light or microwaves has been shown in both cell models. It was proposed that activation of the SAPK/JNK signal pathway plays one of the central roles in cellular stress response to low-power nonionizing radiation.     

Molecular cloning and functional characterization of a prolactin-releasing peptide homolog from Xenopus laevis

Amino acid sequences for identified prolactin (PRL)-releasing peptides (PrRPs) were conserved in mammals (>90%) or teleost fishes (100%), but there were considerable differences between these classes in the sequence (<65%) as well as in the role of PrRP. In species other than fishes and mammals, we have identified frog PrRP. The cDNA encoding Xenopus laevis prepro-PrRP, which can generate putative PrRPs, was cloned and sequenced. Sequences for the coding region showed higher identity with teleost PrRPs than mammalian homologues, but suggested the occurrence of putative PrRPs of 20 and 31 residues as in mammals. The amino acid sequence of PrRP20 was only one residue different from teleost PrRP20, but shared 70% identity with mammalian PrRP20s. In primary cultures of bullfrog (Rana catesbeiana) pituitary cells, Xenopus PrRPs increased prolactin concentrations in culture medium to 130–160% of the control, but PrRPs was much less potent than thyrotropin-releasing hormone (TRH) causing a three- to four-fold increase in prolactin concentrations. PrRP mRNA levels in the developing Xenopus brain peak in early prometamorphosis, different from prolactin levels. PrRP may not be a major prolactin-releasing factor (PRF), at least in adult frogs, as in mammals.     

Presenilin 1 and 2 are expressed differentially in the cerebral cortex of mice during development

Presenilin (PS) 1 and PS2 are multi-pass transmembrane proteins involved in vital brain functions. Studies using transgenic or conditional knockout models show that PS1 is implicated in crucial brain developmental processes. Conversely, PS2 knockout mice do not exhibit any abnormality in the brain morphology, suggesting that PS2 may not be involved in brain development. However, there is no holistic information available for endogenous expression of PS during brain development. Therefore, we have examined the distribution and expression profile of PS1 and PS2 mRNA and protein in the cerebral cortex of prenatal, neonatal and postnatal mice. The results revealed that the distribution and expression profile of PS1 and PS2 mRNA varied significantly in the cerebral cortex during development. In prenatal stages, both PS1 and PS2 mRNA showed high expression at embryonic day (E) 12.5 and downregulation at E18.5. Postnatally, PS1 mRNA showed upregulation from postnatal day 0 (P0) to P45 and thereafter reduction at 20weeks, but PS2 mRNA showed no significant alteration. However, they did not exhibit any significant regional variation except at E18.5, when PS2 showed reduction in temporal and medial temporal lobes as compared to frontal and parietal lobes. Furthermore, PS1 showed significant change in protein expression similar to its mRNA profile. However, PS2 protein expression did not correspond to its mRNA; it was highest at E12.5, downregulated up to P20 and then upregulated at P45 and 20weeks. Taken together, our study demonstrates for the first time that the distribution and expression profile of PS2 is different from PS1 in the mouse cerebral cortex during development.     

Molecular cloning and characterization of a cDNA encoding a novel apoplastic protein preferentially expressed in a shikonin-producing callus strain of Lithospermum erythrorhizon

A cDNA (LEPS-2) encoding a novel cell wall protein was cloned from shikonin-producing callus tissues of Lithospermum erythrorhizon by differential display between a shikonin-producing culture strain and a non-producing strain. The LEPS-2 cDNA encoded a polypeptide of 184 amino acids. The deduced amino acid sequence exhibited no significant homology with known proteins. Expression of LEPS-2 gene as well as accumulation of LEPS-2 protein was highly correlated with shikonin production in L. erythrorhizon cells in culture. In the intact plant, expression of LEPS-2 was detected only in the roots where shikonin pigments accumulated. Cell fractionation experiments and immunocytochemical analysis showed that the protein was localized in the apoplast fraction of the cell walls. The shikonin pigments were also stored on the cell walls as oil droplets. These results indicate that expression of the LEPS-2 is closely linked with shikonin biosynthesis and the LEPS-2 protein may be involved in the intra-cell wall trapping of shikonin pigments.     

Molecular cloning and characterization of a rat A1-adenosine receptor that is widely expressed in brain and spinal cord.

An A1-adenosine receptor has been cloned from a rat brain cDNA library using a probe generated by the polymerase chain reaction. The cDNA encodes a protein of 327 amino acids which is 91% identical to a recently cloned dog A1-adenosine receptor (RDC7). Expression of the rat cDNA in COS-6M and NIH 3T3 cells resulted in ligand binding and functional activity characteristics of an A1-adenosine receptor that is coupled to inhibition of adenylyl cyclase. Examination of the distribution of A1-adenosine receptor mRNA by Northern blot analysis showed that it is highly expressed in brain, spinal cord, testis, and white adipose tissue. In situ hybridization studies revealed an extensive hybridization pattern in the central nervous system, with high levels in cerebral cortex, hippocampus, cerebellum, thalamus, brainstem, and spinal cord. The cloned A1-adenosine receptor may thus mediate many of the modulatory actions of adenosine in neural and endocrine systems.     

绵羊CCNG1基因克隆及表达分析

细胞周期蛋白cyclin G1(CCNG1)是一个重要的细胞周期调控因子,参与哺乳动物颗粒细胞增殖、卵母细胞成熟等繁殖生物学过程,但其在绵羊中鲜有报道。为了研究CCNG1基因对绵羊发情调控以及季节性繁殖的影响,文章对绵羊CCNG1基因进行了克隆和组织表达谱分析,利用Real-time PCR对该基因在多浪羊(常年发情)与中国美利奴羊(季节性繁殖)发情周期不同阶段性腺轴组织的表达变化进行了实时检测。获得了绵羊CCNG1基因部分cDNA序列,其中编码区全长885 bp,编码294个氨基酸。CCNG1蛋白结构经预测存在多个磷酸化位点和蛋白激酶C磷酸化位点。CCNG1基因在所检测绵羊各组织中均有表达,但在卵巢与肾脏中为高丰度表达;CCNG1在不同绵羊品种发情周期不同阶段性腺轴组织的表达变化规律基本一致,卵巢、子宫、松果体、垂体均是在发情期达到峰值。但是在发情期和发情后期卵巢CCNG1的表达量存在显著的品种间差异(P<0.01)。研究结果表明,CCNG1可能通过参与卵泡的生长发育继而达到对绵羊发情和季节性繁殖的调控。