Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2

Neuronal pentraxin 1 (NP1), neuronal pentraxin 2 (NP2), and neuronal pentraxin receptor (NPR) are members of a new family of proteins identified through interaction with a presynaptic snake venom toxin taipoxin. We have proposed that these three neuronal pentraxins represent a novel neuronal uptake pathway that may function during synapse formation and remodeling. We have investigated the mutual interactions of these proteins by characterizing their enrichment on taipoxin affinity columns; by expressing NP1, NP2, and NPR singly and together in Chinese hamster ovary cells; and by generating mice that fail to express NP1. NP1 and NP2 are secreted, exist as higher order multimers (probably pentamers), and interact with taipoxin and taipoxin-associated calcium-binding protein 49 (TCBP49). NPR is expressed on the cell membrane and does not bind taipoxin or TCBP49 by itself, but it can form heteropentamers with NP1 and NP2 that can be released from cell membranes. This is the first demonstration of heteromultimerization of pentraxins and release of a pentraxin complex by proteolysis. These processes are likely to directly effect the localization and function of neuronal pentraxins in neuronal uptake or synapse formation and remodeling.     

NMR studies of nitrophorin distal pocket side chain effects on the heme orientation and seating of NP2 as compared to NP1

The nitrophorins (NP) of the adult blood-sucking insect Rhodnius prolixus fall into two pairs based on sequence identity (NP1,4 (90%) and NP2,3 (79%)), which differ significantly in the size of side chains of residues which contact the heme. These residues include those in the distal pocket of NP2 (I120) and NP1 (T121) and the “belt” that surrounds the heme of NP2 (S40, F42), and NP1(A42, L44). To determine the importance of these residues and others conserved or very similar for the two pairs, including L122(123), L132(133), appropriate mutants of NP2 and NP1 have been prepared and studied by ¹H NMR spectroscopy. Wild-type NP2 has heme orientation ratio (A:B) of 1:8 at equilibrium, while wild-type NP1 has A:B ~ 1:1 at equilibrium. Another difference between NP2 and NP1 is in the heme seating with regard to His57(59). It is found that among the distal pocket residues investigated, the residue most responsible for heme orientation and seating is I120(T121). F42(L44) and L106(F107) may also be important, but must be investigated in greater detail.     

新型冠状病毒N蛋白单克隆抗体制备及双抗体夹心ELISA抗原检测方法的建立

由新型冠状病毒（SARS-CoV-2）感染引起的新型冠状病毒肺炎（COVID-19）自暴发以来,严重威胁着人类健康。建立一种快速、可靠、易操作的可用于SARS-CoV-2感染早期诊断的检测方法,对于疫情防控至关重要。SARS-CoV-2核衣壳蛋白（Nucleocapsid protein,NP）是抗原检测的理想靶点。为了建立一种可快速对SARS-CoV-2 NP进行定量检测的诊断方法,本研究通过免疫山羊制备羊抗SARS-CoV-2多克隆抗体并作为包被抗体,通过杂交瘤细胞技术制备鼠抗NP单克隆抗体并作为检测抗体,建立了双抗体夹心ELISA抗原检测方法。对反应条件进行优化,并验证其线性范围及检测下限、敏感性、特异性、稳定性及准确度。结果显示,建立的方法检测线性范围为1 000 ng/mL～7.8 ng/mL,R2>0.99,检测下限为3.9 ng/mL;敏感性为96.875%,特异性良好,与Vero细胞、SARSCoV-2刺突蛋白、流感病毒等不发生交叉反应;批内和批间变异系数分别为2.1%～11.7%和4.3%～11.8%;检测样品回收率在94.3%～104.8%之间。结果表明,该方...     

Cometabolic biotransformation of nitrobenzene by 3-nitrophenol degrading Pseudomonas putida 2NP8

A strain of Pseudomonas putida (2NP8) capable of growing on both 2-nitrophenol and 3-nitrophenol, but not on nitrobenzene (NB), was isolated from municipal activated sludge. 2-Nitrophenol was degraded by this strain with production of nitrite. Degradation of 3-nitrophenol resulted in the formation of ammonia. Cells grown on 2-nitrophenol did not degrade nitrobenzene. A specific nitrobenzene degradation activity was induced by 3-nitrophenol. Ammonia, nitrosobenzene, and hydroxylaminobenzene have been detected as metabolites of nitrobenzene degradation by cells grown in the presence of 3-nitrophenol. These results indicated a NB cometabolism mediated by 3-nitrophenol nitroreductase.     

The dynamics of the uptake of unionized [NP(NH2)2]2 by barley plants

The dynamics of the uptake by barley plants of trimer phosphonitrile-amide [PN(NH₂)₂]₃ labelled with³²P was studied. It was shown experimentally that the covalent compound [PN(NH₂)₂]₃ enters the plants more rapidly than (NH₄)₂HPO₄. Hence it was concluded that the mechanisms of the transport of [PN(NH₂)₂]₃ and (NH₄)₂HPO₄ into the cells, across the cell membranes, are different.     

禽流感病毒M2e、NP多表位嵌合肽抗原的构建及免疫原性

摘要: 【目的】为了克服传统禽流感疫苗各亚型之间无交叉保护的缺陷,研制抗禽流感通用型疫苗。【方法】 以禽流感病毒M2e、NP表位为基础串联T细胞表位构建4个原核表达载体: pET-3M2e、pET-3M2e-NP1,2-Fc、pET-3M2e-NP1,2、pET-TCE-3M2e-NP1,2。纯化重组蛋白并与弗氏佐剂混合制成疫苗,胸部肌肉注射免疫20日龄非免鸡(150μg/只),4个融合蛋白分为四组,每组十只。ELISA方法检测血清中M2e抗体水平;在MDCK细胞上检测血清与H9N2亚型禽流感病毒的结合能力,在鸡胚上检测其中和能力;以流式细胞仪测定CD4+、CD8+T淋巴细胞的变化。【结果】研究发现,各免疫组均能检测到高水平的ELISA抗体,免疫荧光显示抗血清均能跟病毒特异性结合,中和试验表明抗血清不能中和病毒但能抑制病毒的复制。流式细胞仪检测显示外周血CD4+、CD8+T淋巴细胞所占比例在免疫后均有明显升高(P<0.05),TCE-3M2e-NP1, 2组CD4+、CD8+T淋巴细胞所占比例分别达到47.23%和36.77%,具有细胞免疫的特征。【结论】构建的嵌合肽抗原具有较好的免疫原性,能刺激机体产生体液和细胞免疫,为进一步研制抗禽流感通用型疫苗做出有益的探索。     

Surface Plasmon Assisted Luminescence Enhancement of Ag NP/NWs-Doped SiO2-TiO2-ZrO2:Eu3+ Ternary System

Plasmonics - Non-hydrolytic sol-gel process was employed to prepare Eu3+-doped and silver nanoparticles/nanowires (Ag NP/NWs) co-doped SiO2-TiO2-ZrO2 ternary matrix. XRD, TEM, and SAED analyses...     

Native N-Terminus Nitrophorin 2 from the Kissing Bug: Similarities to and Differences from NP2(D1A)

The first amino acid of mature native nitrophorin 2 is aspartic acid, and when expressed in E. coli, the wild-type gene of the mature protein retains the methionine-0, which is produced by translation of the start codon. This form of NP2, (M0)NP2, has been found to have different properties from its D1A mutant, for which the Met0 is cleaved by the methionine aminopeptidase of E. coli (R.?E. Berry, T.?K. Shokhireva, I. Filippov, M.?N. Shokhirev, H. Zhang, F.?A. Walker, Biochemistry 2007, 46, 6830). Native N-terminus nitrophorin 2 ((ΔM0)NP2) has been prepared by employing periplasmic expression of NP2 in E. coli using the pelB leader sequence from Erwinia carotovora, which is present in the pET-26b expression plasmid (Novagen). This paper details the similarities and differences between the three different N-terminal forms of nitrophorin 2, (M0)NP2, NP2(D1A), and (ΔM0)NP2. It is found that the NMR spectra of high- and low-spin (ΔM0)NP2 are essentially identical to those of NP2(D1A), but the rate and equilibrium constants for histamine and NO dissociation/association of the two are different.     

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein(NP) and extracellular domain of matrix protein 2(M2e) genes from the influenza A virus A/Beijing/30/95(H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34(PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP-and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD_(50) of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.     

抗汉坦病毒NP scFv基因表达及生物活性分析

诱导已构建的重组质粒pGEX-6P—1—scFv原核表达抗汉坦病毒核衣壳蛋白单链抗体,并用酶免疫实验检测单链抗体生物活性。用IPTG诱导重组原核表达质粒pGEX-6P-1-scFv表达抗汉坦病毒NP单链抗体融合蛋白,经亲和层析纯化,并应用SDS—PAGE电泳检测单链抗体融合蛋白,应用酶免疫实验检测抗NP单链抗体生物学活性。SDS—PAGE电泳检测显示,原核重组质粒pGEX-6P-1-scFv已表达分子量约为56ku的单链抗体融合蛋白;酶免疫实验检测显示,单链抗体具有与汉坦病毒NP抗原特异性结合的生物学活性。结果表明,已构建的原核表达重组质粒pGEX-6P-1-scFv,能够成功表达具有与汉坦病毒NP抗原特异性结合生物学活性的单链抗体。     

Biotransformation of hydroxylaminobenzene and aminophenol by Pseudomonas putida 2NP8 cells grown in the presence of 3-nitrophenol

Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated.     

Biotransformation of Hydroxylaminobenzene and Aminophenol by Pseudomonas putida 2NP8 Cells Grown in the Presence of 3-Nitrophenol

Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated.     

A neuronal protein (NP185) associated with clathrin-coated vesicles. Characterization of NP185 with monoclonal antibodies

Two monoclonal antibodies (S-8G8 and S-6G7) are characterized that react with an abundant neuronal protein associated with brain clathrin-coated vesicles (CCVs). This 185-kDa polypeptide (NP185) is not a transmembrane cargo molecule and is distinguishable from clathrin by several criteria including neuronal specificity, chymotryptic sensitivity, migration during two-dimensional gel electrophoresis, lack of cross-reactivity of S-8G8 or S-6G7 with purified clathrin, and lack of associated clathrin light chains. When 0.9 M NaCl extracts of CCVs were diluted and immunoprecipitated by either S-8G8 or S-6G7, NP185 precipitated as a complex with a fraction of the CCV assembly polypeptides. Immunofluorescence microscopy of PC12 cells cultured in nerve growth factor (NGF) revealed that NP185 was distributed in a punctate manner throughout the mature neurites. Immunoblot analysis of PC12 cell extracts, taken at various times during NGF-induced differentiation, revealed that steady-state accumulation of NP185 reaches significant levels 3 days after the addition of NGF and returns to undetectable levels when NGF is removed from the cultures. Significantly, the quantity of NP185 detected in differentiated PC12 cells exceeded the quantity of clathrin. These data indicate that while NP185 may be a specialized component of neuronal CCVs, its function in neuronal cells cannot be associated exclusively with these organelles.     

溶藻菌株NP23的紫外诱变选育

应用紫外诱变技术对溶藻菌株NP23进行紫外诱变处理。经过粗筛后,从8株诱变株中选出2株对绿藻中小球藻和蓝藻中惠氏微囊藻的去除效果明显优于原始菌株的突变株NP23-1和NP23-4,其溶藻率(叶绿素a的去除率)比原始菌株提高30%-35%。连续6代测试,2株诱变菌株NP23-1和NP23-4溶藻率都很稳定,表明所得突变株是比原始菌株更优秀的溶藻菌株。     

Correction to: Surface Plasmon Assisted Luminescence Enhancement of Ag NP/NWs-Doped SiO2-TiO2-ZrO2:Eu3+ Ternary System

Plasmonics - The original version of this article unfortunately contained a mistake.     

Ubiquitination and deubiquitination of NP protein regulates influenza A virus RNA replication

Influenza A virus RNA replication requires an intricate regulatory network involving viral and cellular proteins. In this study, we examined the roles of cellular ubiquitinating/deubiquitinating enzymes (DUBs). We observed that downregulation of a cellular deubiquitinating enzyme USP11 resulted in enhanced virus production, suggesting that USP11 could inhibit influenza virus replication. Conversely, overexpression of USP11 specifically inhibited viral genomic RNA replication, and this inhibition required the deubiquitinase activity. Furthermore, we showed that USP11 interacted with PB2, PA, and NP of viral RNA replication complex, and that NP is a monoubiquitinated protein and can be deubiquitinated by USP11 in vivo. Finally, we identified K184 as the ubiquitination site on NP and this residue is crucial for virus RNA replication. We propose that ubiquitination/deubiquitination of NP can be manipulated for antiviral therapeutic purposes.