Na-K-2Cl共同转运蛋白(NKCC2)基因研究进展

Na-K-2Cl共同转运体在维持肾小管髓袢升支粗段对NaCl 的重吸收,机体容量控制方面起重要作用,NKCC2基因突变可使其编码蛋白质结构、功能改变, 使体内血压调节的稳态发生变化。     

GAD67-GFP基因敲入小鼠黑质网状部神经元GABA与氯离子转运体KCC2和NKCC1的共存

为了观察谷氨酸脱羧酶67-绿色荧光蛋白(GAD67-GFP)基因敲入小鼠黑质网状部(SNr)内,表达GFP的GABA能神经元与一对功能相反的Cl-共转运体(K+-Cl-cotransporter2,KCC2;Na+-K+-Cl-cotransporter1,NKCC1)的共存情况,本研究分别运用原位分子杂交与免疫组织化学相结合以及GFP与KCC2或NKCC1免疫荧光染色相结合的双重标记方法,在光学显微镜和激光共聚焦显微镜下同时进行观察。结果显示:(1)SNr内95%以上的GFP阳性神经元同时表达KCC2 mRNA,而50%表达KCC2 mRNA的阳性神经元呈GFP阳性;(2)SNr内80%以上的GFP阳性神经元同时表达NKCC1 mRNA,约35%表达NKCC1 mRNA的阳性神经元呈GFP阳性;(3)SNr内90%以上的GFP阳性神经元同时表达KCC2,双标神经元约占KCC2阳性神经元的50.5%;(4)SNr内80.5%以上的GFP阳性神经元同时表达NKCC1,双标神经元约占NKCC1阳性神经元的42.5%。以上结果表明,SNr内表达GFP的GABA能神经元大部分与KCC2和NKCC1共存,提示氯离子共转运体可能对SNr内GABA能神经元起重要的调控作用。     

Novel neuronal proteolipid protein isoforms encoded by the human myelin proteolipid protein 1 gene

The human myelin proteolipid protein 1 gene (hPLP1), which encodes the major structural myelin proteins of the central nervous system (CNS), is classically described as expressed in the oligodendrocytes, the CNS myelinating cells. We identified two new exons in the intron 1 of the hPLP1 gene that lead to the expression of additional mRNA and protein isoforms mainly expressed in neurons instead of oligodendrocytes. Those novel neuronal PLP isoforms are detected as soon as human fetal development and their concomitant expression is specific of the human species. As classical PLP proteins, the novel protein isoforms seem to be addressed to the plasma membrane. These results suggest for the first time that PLP may have functions in humans not only in oligodendrocytes but also in neurons and could be implicated in axono-glial communication. Moreover, this neuronal expression of the hPLP1 gene might explain the neuronal dysfunctions in patients carrying hPLP1 gene mutations.     

NKCC1和KCC2在成年大鼠大脑皮质神经元的胞体和树突的不同表达

γ-氨基丁酸(GABA)是成年哺乳动物脑内主要的抑制性神经递质,但电生理的研究表明,GABA在成熟皮质神经元的树突部位可以产生兴奋性作用,但该现象的形态学基础,目前尚不清楚。GABA产生兴奋性作用的关键主要依赖于神经元胞内的氯离子浓度,其中Na+-K+-Cl-共转运体1(NKCC1)促进细胞内Cl-堆积,而K+-Cl-共转运体2(KCC2)则外排胞内的Cl-,降低胞内的Cl-浓度。本研究应用免疫荧光组织化学双重标记结合荧光强度分析,检测NKCC1和KCC2在成年大鼠脑皮质和培养的大鼠脑皮质神经元树突和胞体的表达和分布情况。结果显示:成年大鼠皮质神经元的胞浆和细胞膜均有NKCC1的表达,而KCC2主要表达在神经元胞体和树突膜上,其中NKCC1在神经元树突上的表达水平比胞体高,而KCC2的表达水平在树突和胞体膜上没有明显差异。皮质神经元经培养20d后,NKCC1和KCC2在树突和胞体的表达模式与在体的分布相类似。本研究结果提示,NKCC1在大鼠皮质神经元树突的表达较多,可能是GABA兴奋神经元树突的原因。     

Phenotypic and functional characterization of human CD25+ B cells

We demonstrate that humans have a phenotypically and functionally distinct subset of B lymphocytes that express the interleukin (IL)-2 receptor (IL-2R) alpha-chain, cluster of differentiation (CD) 25. We found that one-third of the circulating CD20+ B cells expressed CD25 and, using fluorescence-activated cell sorter (FACS) analysis, that these cells were significantly larger and more granulated than B cells not expressing CD25. The simultaneous expression of the other two subunits (CD122 and CD132) and the proliferative responses of cells expressing CD25 to IL-2 suggested that, in addition to CD25, functional IL-2 receptors were expressed on this cell population. CD25 expression on B cells was selectively up-regulated by Toll-like receptor 2 (TLR2), TLR4, and TLR9 ligands but not by a TLR3 ligand or Epstein-Barr virus (EBV) stimulation. Blockade of the nuclear factor (NF)-kappaB pathway completely abolished CD25 up-regulation by these B cells. Interestingly, CD25+ B cells expressed significantly higher levels of surface immunoglobulins but lacked the ability to secrete immunoglobulin (Ig), as compared with CD25- B cells. Furthermore, CD25+ B cells performed significantly better as antigen-presenting cells in allogeneic mixed lymphocyte reactions (MLR), which may be a result of their expression of high levels of the costimulatory molecules CD27 and CD80. Finally, blocking of CD25 on B cells led to an almost total abrogation of MLR. Our results indicate that CD25+ B cells have distinct phenotypic and functional properties, including the ability to contribute to antigen presentation, which is linked to their expression of CD25. Finally, the differential regulation of CD25 expression via selective TLR ligands suggests a role for CD25+ B cells in bridging innate and acquired immune responses.     

In silico studies on the comparative characterization of the interactions of SARS-CoV-2 spike glycoprotein with ACE-2 receptor homologs and human TLRs

Coronavirus disease-2019 (COVID-19) outbreak due to novel coronavirus or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has come out as a major threat for mankind in recent times. It is continually taking an enormous toll on mankind by means of increasing number of deaths, associated comorbidities, and socioeconomic loss around the globe. Unavailability of chemotherapeutics/vaccine has posed tremendous challenges to scientists and doctors for developing an urgent therapeutic strategy. In this connection, the present in silico study aims to understand the sequence divergence of spike protein (the major infective protein of SARS-CoV-2), its mode of interaction with the angiotensin-converting enzyme-2 receptor (ACE2) receptor of human and related animal hosts/reservoir. Moreover, the involvement of the human Toll-like receptors (TLRs) against the spike protein has also been demonstrated. Our data indicated that the spike glycoprotein of SARS-CoV-2 is phylogenetically close to bat coronavirus and strongly binds with ACE2 receptor protein from both human and bat origin. We have also found that cell surface TLRs, especially TLR4 is most likely to be involved in recognizing molecular patterns from SARS-CoV-2 to induce inflammatory responses. The present study supported the zoonotic origin of SARS-CoV-2 from a bat and also revealed that TLR4 may have a crucial role in the virus-induced inflammatory consequences associated with COVID-19. Therefore, selective targeting of TLR4-spike protein interaction by designing competitive TLR4-antagonists could pave a new way to treat COVID-19. Finally, this study is expected to improve our understanding on the immunobiology of SARS-CoV-2 and could be useful in adopting spike protein, ACE2, or TLR-guided intervention strategy against COVID-19 shortly.     

Kinetics and functional characterization of the glycine receptor α2 and α3 subunit

In recent studies one serine residue (Ser-346) within the protein-kinase-A (R-E-S-R) consensus sequence of the GlyRα3 intracellular loop has proven to be an essential target for prostaglandin-E₂-mediated phosphorylation, which further modulates spinal nociceptive transmission and central inflammatory pain sensitization. In the present study we investigated the effect of Ser-346 phosphorylation and Ser-346 mutation on receptor kinetics and function using whole-cell patch-clamp recordings in transfected HEK 293 T cells. We compared biophysical properties of wild type GlyRα3 and two site-directed mutants, where Ser-346 was replaced by alanine or aspartate, in the absence and presence of prostaglandin-E₂. The mutation to alanine was accompanied by significantly altered dose–response and desensitization properties. Mutation to aspartate had only minor effects on receptor kinetics and function. Phosphorylation of Ser-346 slowed desensitization and decreased glycinergic currents in GlyRα3/mEP2 transfected cells. In addition, we demonstrated that prostaglandin-E₂ also had an effect on the GlyRα2 subunit. Exposure to prostaglandin-E₂ decreased the maximum peak current amplitude of glycinergic currents in GlyRα2/mEP2 transfected cells in the same manner as phosphorylation of the GlyRα3 subunit. It led to a significant increase of the desensitization time constants and thus significantly affected the desensitization behaviour. These results indicate that the GlyRα2 and the GlyRα3 subunits act as important subunits for the modulation of glycine receptor kinetics and function.     

Assessment of the functional impact on the pre‐mRNA splicing process of 28 nucleotide variants associated with Pompe disease in GAA exon 2 and their recovery using antisense technology

Glycogen storage disease II (GSDII), also called Pompe disease, is an autosomal recessive inherited disease caused by a defect in glycogen metabolism due to the deficiency of the enzyme acid alpha‐glucosidase (GAA) responsible for its degradation. So far, more than 500 sequence variants of the GAA gene have been reported but their possible involvement on the pre‐messenger RNA splicing mechanism has not been extensively studied. In this work, we have investigated, by an in vitro functional assay, all putative splicing variants within GAA exon 2 and flanking introns. Our results show that many variants falling in the canonical splice site or the exon can induce GAA exon 2 skipping. In these cases, therefore, therapeutic strategies aimed at restoring protein folding of partially active mutated GAA proteins might not be sufficient. Regarding this issue, we have tested the effect of antisense oligonucleotides (AMOs) that were previously shown capable of rescuing splicing misregulation caused by the common c.‐32‐13T>G variant associated with the childhood/adult phenotype of GSDII. Interestingly, our results show that these AMOs are also quite effective in rescuing the splicing impairment of several exonic splicing variants, thus widening the potential use of these effectors for GSDII treatment.     

神经母细胞瘤SK-N-SH细胞内DHRS4L2基因一种新选择性剪接亚型S4的克隆和亚细胞定位

目的 神经母细胞瘤SK-N-SH细胞系脱氢酶/还原酶(SDR家族)成员4类2[dehydrogenase/reductase(SDR family)member 4 like 2,DHRS4L2]基因的一种新的选择性剪接亚型克隆、生物信息学分析及其亚细胞定位.方法 以SK-N-SH细胞cDNA为模板,PCR扩增DHRS4基因簇Ea1转录本.将PCR产物A-T克隆至pGEMT-Easy质粒,对质粒进行DNA Sanger测序.将测序所得序列用NCBI ORF finder分析其编码区,用Motif Scan分析预测蛋白氨基酸序列.用Clustal Omega进行蛋白序列比对分析.将新亚型完整编码框cDNA以及删除偶核定位信号的编码框分别插入pEGFP-C1质粒,所得质粒和空质粒分别转染SK-N-SH细胞,在荧光显微镜下观察转染表达蛋白亚细胞定位.结果 用RT-PCR和Sanger测序方法发现,SK-N-SH表达DHRS4L2 Ea1转录本,未检测到其表达脱氢酶/还原酶(SDR家族)成员4类1[dehydrogenase/reductase(SDR family)member 4 like 1,DHRS4L1]的Ea2转录本.DHRS4L2 Ea1表达一个新的选择性剪接亚型DHRS4L2-S4(KU141377),由AY616183基础上在Ea1与E2外显子之间插入新外显子Ej形成,外显子Ej含有新亚型翻译起始密码子ATG.转录本KU141377预测蛋白羧基端具有偶核定位信号(bipartite nuclear localization signal,NLS),提示其可能定位于细胞核.绿色荧光蛋白融合蛋白实验显示,在SK-N-SH细胞该蛋白定位于细胞核.该蛋白还含有一个甘氨酸密集区(glycine-rich region)和阿片样生长因子受体重复(opioid growth factor receptor repeat)序列.结论 研究发现SK-N-SH细胞表达的一种DHRS4L2新选择性剪接亚型KU141377,其预测编码蛋白含有细胞偶核定位信号,融合荧光蛋白实验显示该新亚型定位于细胞核,这为后续研究DHRS4L2在神经母细胞瘤中的潜在功能奠定基础.     

Molecular characterization of a novel fission yeast gene spUAP2 that interacts with the splicing factor spU2AF59

A protein essential for pre-mRNA splicing, the U2 auxiliary factor (U2AF), is composed of a large and small subunit. Previously we cloned and characterized both subunits, spU2AF⁵⁹ and spU2AF²³, from fission yeast. We now report a novel U2AF-associated-protein, spUAP2, which interacts with both subunits. SpUAP2 contains a classical and a degenerate RNA recognition motif (RRM), both of which are required for interaction with spU2AF⁵⁹. Interaction also requires the arginine/serine-rich region and the first RRM of spU2AF⁵⁹. A null allele of the gene for spUAP2 is lethal. Received: 23 May / Accepted: 6 August 1997     

Carbohydrate heterogeneity of human myeloma proteins of the IgA1 and IgA2 subclasses

Comparative studies of the N-linked carbohydrate chains of human myeloma proteins of the IgA1 and IgA2 subclasses were performed. The N-linked carbohydrate chains were released by hydrazinolysis from the polypeptide backbone, converted to radioactive oligosaccharides by sodium borotritide reduction after N-acetylation and separated into one neutral and two acidic fractions by paper electrophoresis. The acidic oligosaccharides were completely converted to neutral oligosaccharides by sialidase treatment, indicating that they were sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were further fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed that human myeloma IgA proteins contained significant amounts of biantennary complex-type carbohydrate chains in addition to a small amount of the high mannose-type. The results indicated that the oligosaccharide structures of human IgA1 and IgA2 display a high degree of heterogeneity not only in the number of carbohydrate chains, but also in their composition.     

Phenotypic characterization of regulatory T cells in the human decidua

Pregnancy is a unique situation for the maternal immune system. We have studied and identified a CD4+CD25+ regulatory T (Treg) cell population isolated from the human decidua. This mucosal surface in the uterus is in direct contact with semiallogenic fetal cells. We observed that about 14% of the decidual CD4+ T cells have the CD4+CD25+ phenotype. The decidual CD4+CD25+ T cells expressed high frequency of intracellular CTLA-4 (CTLA-4i). The majority of CD4+CD25+CTLA-4i+ cells were also positive for GITR and OX40, typical markers for human Treg cells. The frequency of CD4+CD25+ T cells in the peripheral blood from pregnant women was found to be increased during the first and second trimester of gestation when compared to nonpregnant controls. Being an important molecule for Treg cells, the role of CTLA-4 in the regulation of indoleamine 2,3-dioxygenase (IDO) expression was also examined. The stimulation with CTLA-4Ig did not increase IDO mRNA expression in CD14+ cells from pregnant women, while IFN-gamma was observed to up-regulate IDO expression. The presence of Treg cells in the human decidua suggests that these cells are important in protecting the fetus from alloreactive immune responses at the maternal-fetal interface.     

Localization of metallothionein in aged human brain

The localization of metallothionein (MT), a small molecular weight heavy metal binding protein in aged human brain, was investigated by immunohistochemical techniques. The amount of MT and heavy metals (Zn, Cu) were also assayed by radioimmunoassay and atomic absorption spectrophotometry, respectively. Immunohistochemically, MT was found in the pia mater, ependymal cells, protoplasmic astrocytes nand glial processes neuropil of the gray matter and fibrous astrocytes of the white matter of the telencephalon, whereas oligodendroglia and microglia did not show any positive immunostaining for MT. Cytoplasm, glial processes and some nuclei of protoplasmic and fibrous astrocytes showed strong MT immunostaining. Vascular feet and adventitia were also positive for MT immunostaining. Moreover, the pia mater, astrocytes and ependyma in the diencephalon, mesencephalon, pons, medulla oblongata and spinal cord showed the same positive immunostaining for MT as the telencephalon. In the cerebellum, Bergmann's glia, protoplasmic astrocytes of the granular layer and fibrous astrocytes of the white matter showed strongly positive immunostaining for MT.
According to the radioimmunoassay, the amount of MT in the brain was relatively high at about 39.3 ± 9.5 μg/g wet weight; of the heavy metals in the brain, zinc and copper were detected.     

Splicing mutations at the HPRT locus in human T-lymphocytes in vivo

We studied 58 splicing mutations originating in vivo at the hypoxanthine guanine phosphoribosyltransferase (HPRT) locus in T-cells of 30 nonsmoking males. A nonrandom distribution of skipped exons was seen after cDNA sequence analysis, with 71% involving exons 2–3 (15), 4 (11), and 8 (15). The mutations likely to have caused the aberrant splicing were identified in 36 mutants by genomic sequencing. The most frequently observed mutations were simple base substitutions (27) and small deletions (7). Among the base substitutions, 23 occurred in the splice consensus sequences, mainly at the highly conserved dinucleotides (21), and preferentially in the acceptor sites (15). The remaining four base substitutions occurred in the coding sequence where one tandem base substitution, one single bp insertion, and two single bp deletions were also observed. The predicted change in three of the base substitutions would be a stop codon. The tandem mutation (CC → TT) occurred at position 550–551, a possible hotspot for splicing mutations (five of nine previously reported base substitutions at position 551, all C → T, resulted in abnormal splicing). Four of the base substitutions were new HPRT mutations, two in splice sites (IVS7-3T → G and IVS8 + 3A → C) and two in the coding sequence (307A → T and 594C → G). All the small deletions (>1 bp) affected the acceptor sites. The only three identified mutations related to skipping of exons 2 and 3 were located within exon 3, suggesting a frequent involvement of unknown splicing elements distant from these exons. Environ. Mol. Mutagen. 32:25–32, 1998 © 1998 Wiley-Liss, Inc.