Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or β-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a–C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.     

Phosphocalyculin C as a pyrophosphate protoxin of calyculin C in the marine sponge Discodermia calyx

Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C.     

SVCT与维生素C内稳态的保持

维生素C(又名抗坏血酸)是一种基本的微量营养素,作为辅助因子参与多个酶促反应,同时还是一种自由基清除剂。维生素C内稳态主要由两种钠离子依赖的维生素C转运蛋白(sodium-dependent vitamin C transporter,SVCT)——SVCT1和SVCT2来保持。SVCT1在内皮系统表达,介导了维生素C的肠吸收和肾脏重吸收;而SVCT2表达广泛,表达于脑、骨骼和其他组织,保护这些组织免遭氧化损伤。SVCT的遗传多态性与癌症的发生密切相关。对SVCT介导的维生素C内稳态的保持机制的研究,可使维生素C更好地应用于临床。     

Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor

Zinc is an essential trace element for human nutrition and is critical to the structure, stability, and function of many proteins. Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation but down-regulate the extrinsic pathway of coagulation. The protein C pathway plays a key role in blood coagulation and inflammation. At present there is no information on whether zinc modulates the protein C pathway. In the present study we found that Zn²⁺ enhanced the binding of protein C/activated protein C (APC) to endothelial cell protein C receptor (EPCR) on endothelial cells. Binding kinetics revealed that Zn²⁺ increased the binding affinities of protein C/APC to EPCR. Equilibrium dialysis with ⁶⁵Zn²⁺ revealed that Zn²⁺ bound to the Gla domain as well as sites outside of the Gla domain of protein C/APC. Intrinsic fluorescence measurements suggested that Zn²⁺ binding induces conformational changes in protein C/APC. Zn²⁺ binding to APC inhibited the amidolytic activity of APC, but the inhibition was reversed by Ca²⁺. Zn²⁺ increased the rate of APC generation on endothelial cells in the presence of physiological concentrations of Ca²⁺ but did not further enhance increased APC generation obtained in the presence of physiological concentrations of Mg²⁺ with Ca²⁺. Zn²⁺ had no effect on the anticoagulant activity of APC. Zn²⁺ enhanced APC-mediated activation of protease activated receptor 1 and p44/42 MAPK. Overall, our data show that Zn²⁺ binds to protein C/APC, which results in conformational changes in protein C/APC that favor their binding to EPCR.     

X-ray crystal structure of the C4d fragment of human complement component C4

C4 fulfills a vital role in the propagation of the classical and lectin pathways of the complement system. Although there are no reports to date of a C4 functional activity that is mediated solely by the C4d region, evidence clearly points to it having a vital role in a number of the properties of native C4 and its major activation fragment, C4b. Contained within the C4d region are the thioester-forming residues, the four isotype-specific residues controlling the C4A/C4B transacylation preferences, a binding site for nascent C3b important in assembling the classical pathway C5 convertase and determinants for the Chido/Rodgers (Ch/Rg) blood group antigens. In view of its functional importance, we undertook to determine the three-dimensional structure of C4d by X-ray crystallography. Here we report the 2.3A resolution structure of C4Ad, the C4d fragment derived from the human C4A isotype. Although the approximately 30% sequence identity between C4Ad and the corresponding fragment of C3 might be expected to establish a general fold similarity between the two molecules, C4Ad in fact displays a fold that is essentially superimposable on the structure of C3d. By contrast, the electrostatic characteristics of the various faces of the C4Ad molecule show marked differences from the corresponding faces of C3d, likely reflecting the differences in function between C3 and C4. Residues previously predicted to form the major Ch/Rg epitopes were proximately located and accessible on the concave surface of C4Ad. In addition to providing further insights on the current models for the covalent binding reaction, the C4Ad structure allows one to rationalize why C4d is not a ligand for complement receptor 2. Finally the structure allows for the visualization of the face of the molecule containing the binding site for C3b utilized in the assembly of classical pathway C5 convertase.     

人Sema4C重组腺病毒载体的构建及其在小鼠成肌细胞系C2C12中的表达

利用Ad5腺病毒载体系统构建人Sema4C基因重组腺病毒表达载体并在成肌细胞系C2C12中表达,并初步探讨Sema4C基因在成肌发育过程中的可能作用。利用脂质体介导重组腺病毒载体转染HEK293细胞,包装出完整的腺病毒;将重组腺病毒载体感染C2C12成肌细胞后,利用激光共聚焦显微镜观察发现12h即有绿色荧光表达,24h后绿色荧光蛋白表达最强;流式细胞仪检测病毒的感染效率几乎达100%。WB检测结果表明感染重组腺病毒载体组C2C12细胞Sema4C蛋白的表达量明显高于空载体对照组（P<0.01）。为了进一步观察Sema4C基因对C2C12细胞增殖分化的影响,流式细胞仪检测了病毒感染48h后C2C12细胞的增殖指数,并对感染后诱导分化的C2C12细胞的分化情况进行了观察。我们的结果首次表明,过表达外源性人Sema4C基因不仅能使C2C12细胞的G0/G1期比例增加,细胞的增殖指数下降,同时在分化培养条件下还能促进C2C12细胞肌管的形成。     

Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells

Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The protein serves as a marker for early stages in chondrogenesis and T-cell development. Itm2A is also highly expressed in skeletal muscle. In order to understand the role of Itm2A in muscle development, we constitutively overexpressed exogenous Itm2A in C2C12 myoblast cells. Several clones expressing high levels of Itm2a were isolated and characterized. Overexpression was associated with enhanced tube formation and the appearance of multinuclear cells. Gene expression analysis demonstrated that muscle creatin kinase was upregulated in the presence of exogenous Itm2A. Interestingly, proliferation rates were not altered in the undifferentiated myoblast C2C12 cells. These results demonstrate that overexpression of Itm2a in C2C12 enhances myogenic differentiation in vitro.     

C3植物中C4途径的研究进展

综述了Ｃ３植物中Ｃ４途径的发现及研究现状：阐述了Ｃ３植物中Ｃ４途径的几种作用机理;根据Ｃ３植物中Ｃ４途径的存在,探讨了改造Ｃ３植物的遗传特性;并展望了这一领域的研究前景。     

C型肉毒梭菌毒素的稳定性研究

报道了保存肉毒毒素经常会遇到的某些因素对Ｃ型肉毒梭菌Ｃ６５１４培养滤液稳定性的影响观察结果。结果表明,提高环境温度及介质酸碱度、日光照射、激烈震荡都能使毒素的毒力下降。甘油或明胶磷酸盐缓冲剂对于保存Ｃ型肉毒毒素都是较好的活性稳定剂。含甘油或明胶磷酸盐缓冲剂的毒素在４℃下保存１２个月后,毒力的变动甚微。此二法都不需要特殊设备,材料易得,操作简便,比较实用。     

Genetic transformation of the C3–C4 intermediate plant, Flaveria pubescens (Asteraceae)

The dicot genus Flaveria (Asteraceae), besides species with C₃ or C₄ photosynthesis, contains taxa with a broad range of different states of transition between the two major photosynthetic types. We have developed a reproducible and efficient Agrobacterium-mediated method for the stable genetic transformation of the C₃–C₄ intermediate species F. pubescens. Fusion constructs of the reporter gene β-glucuronidase (uidA, GUS) to several plant promoters, mainly derived from genes encoding subunits of the glycine cleavage system (gdcs), have been used to confirm the reproducibility and efficiency of the method. The stable integration of the foreign DNA has been examined by Southern analysis, kanamycin resistance, GUS enzyme activities and histochemical staining. Transformed shoots can be routinely obtained within 8–10 weeks after co-cultivation with A. tumefaciens. Received: 16 April 1996 / Revision received: 12 July 1996 / Accepted: 28 July 1996     

Linkage between the CYP2C8 and CYP2C9 genetic polymorphisms

Cytochrome P450 (CYP) 2C8 and 2C9 are polymorphic enzymes. The CYP2C8*3 and CYP2C9*2 are the major variant alleles in Caucasian populations. The enzymes encoded by these variant alleles have impaired function for the metabolism of several drug substrates. In the present study 1468 subjects that were used as population-based controls in the Stockholm Heart Epidemiology Program (SHEEP) were genotyped by allelic discrimination using a 5'-nuclease assay for CYP2C8*1, 2C8*3, 2C9*1, 2C9*2, and 2C9*3 variant alleles in which the frequencies appeared to be 0.91, 0.095, 0.83, 0.11, and 0.066, respectively. Approximately, 96% of the subjects with CYP2C8*3 allele also carried a CYP2C9*2 and 85% of the subjects that had CYP2C9*2 variant also carried a CYP2C8*3. The number of subjects carrying both of the CYP2C8*1*3 and CYP2C9*1*2 was 4.5-fold higher than expected. This strong association may be of importance especially for the metabolism of common substrates of CYP2C8 and CYP2C9 like arachidonic acid that produces physiologically active metabolites.     

Isotyping of component C4 of human complement using differences in the functional activity of isotypes C4A and C4B

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated on activators sorbed on ELISA microplates: immunoglobulin IgG3 or liposaccharide of theShigella sonnei cell walls, which activates the complement by binding component C1. The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated with liposaccharide, isotype C4B is determined. The radio of the activities determined by the two methods indicates the deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

海北高寒草甸土壤有机碳同位素组成及C3/C4碳源的变化

通过对高寒嵩草草甸土壤剖面不同深度(0～5cm,5～15cm,15～25cm,25～35cm,35～50cm,50～65cm)有机碳稳定性碳同位素的测定发现,土壤有机碳稳定性同位素(δ^13C)随土壤深度的增加而变大。表层土壤(0～5cm,定义为现代土壤)的δ^13C值最小,基本上接近现代植被的碳同位素特征。在土层5～10cm深度以下(粗略地定为古土壤),土壤有机碳稳定性同位素骤然上升,与表层土壤的同位素特征明显不同。考虑到影响土壤碳同位素的诸多因素,通过稳定性碳同位素的质量平衡模型计算,得出初步结果：来自C4(或CAM)植物的碳源随土壤深度的增加而增大。进一步推测,该地区植被可能经历由C4植物占优势的群落向C3植物占优势的群溶演化的过程。在这个过程中,大气碳同位素的变化和土壤有机质的形成过程(有机质淋溶过程)等也会引起土壤碳同位素的升高,因此质量平衡模型可能会过多地估算C4组分,而低估C3组分。     

脉冲电磁场对C2C12 成肌细胞增殖影响的实验研究

目的:研究不同强度脉冲电磁场干预对成肌细胞增殖的影响。方法:10Hz脉冲低频电磁场刺激经复苏后培养贴壁良好的C2C12成肌细胞,根据不同磁场强度和作用时间将其分为A、B、C组,无磁场干预的为对照组。采用RT-q PCR检测不同磁场强度下成肌细胞标记基因Myf5、Myo D及Pax7的m RNA的表达。结果:经RT-q PCR检测三种基因的表达情况,Myf5 m RNA在1.5 m T磁场强度下照射第五天表达最高;0.5 m T磁场强度下Myf5 m RNA的表达与对照组相比无统计学意义(P>0.05);1.0 m T磁场强度下Myf5 m RNA表达与对照组比较差异具有统计学意义(P<0.05);1.5 m T磁场强度下Myf5 m RNA表达与对照组比较差异具有统计学意义(P<0.05)。对照组Myo D m RNA的表达要比磁场作用下表达要高。三个磁场强度下Myo D m RNA表达与对照组相比均无统计学意义(P>0.05)。0.5 m T、1.0 m T磁场强度下Pax7 m RNA的表达要比对照组要高,与对照组相比具有统计学意义(P<0.05);1.5 m T磁场强度下Pax7 m RNA表达与对照组相比无统计学意义(P>0.05)。结论 :1.5 m T脉冲电磁场强度下作用5天对体外培养的成肌细胞Myf5 m RNA标记基因增殖促进作用最强。     

Generation of an antibody with a designed specificity difference for protein C and activated protein C

Rabbit polyclonal antibodies to a synthetic peptide, NH₂-Asp-Thr-Asn-Gln-Val-Asp-Gln-Lys-Asp-Gln-Leu-Asp-Phe-Arg-CONH₂ (APep), have been produced. This sequence is identical to that contained in the tetradecapeptide released from bovine protein C (PC) as a result of its conversion to its activated form (APC), except that Phe₁₃ replaced the normal Pro₁₃, in order to discourage cross-reactivity of antibodies to the carboxylterminal portion of APep with PC. The antibody pool obtained reacted with PC and showed virtually no cross-reactivity toward either APC or several typical plasma proteins. This general approach should serve well as a means of production of antibodies with a designed specificity capable of distinguishing between forms of the same protein that arise by release of peptide material.     

Adiponectin binds C1q and activates the classical pathway of complement

The adipose-specific protein adiponectin binds to a number of target molecules, including damaged endothelium and the surface of apoptotic cells. However, the significance of this binding remains unclear. This study demonstrates the binding of purified C1q to recombinant adiponectin under physiological conditions, and the dependence of this upon Ca⁺⁺ and Mg⁺⁺. Binding was enhanced by metaperiodate-mediated destruction of glucosylgalactosyl sugars on adiponectin. Adiponectin was bound by the globular domain of the A chain of collagenase-digested C1q, and C1q binding induced deposition of C4 and C3 through activation of the classical complement pathway. After Western blotting, affinity-purified adiponectin from human serum bound C1q, whereas adiponectin in whole serum did not, unless pre-treated with metaperiodate. These results suggest adiponectin is member of the pattern-recognition family of defence collagens, able to bind target molecules and activate complement. It may therefore play an important role in innate immunity and autoimmune phenomena.     

用T-A载体克隆技术构建丙肝病毒C+E1区真核表达质粒pSVL-HCV/C+E1

用ＢａｍＨＩ和ＨｉｎｄＩＩ将丙肝病毒Ｃ＋Ｅ１ＤＮＡ片段从其克隆载体ｐＧＥＭ３ｚｆ－ＨＣＶ／Ｃ＋Ｅ１上切下,经Ｔａｑ酶补齐３’末端后插入到载体ｐＳＶＬ－Ｔ中,构建成丙肝病毒Ｃ＋Ｅ１真核表达载体ｐＳＶＬ－ＨＣＶ／Ｃ＋Ｅ１。本实验中重组效率达６４．７％（１１／１７）,正向插入为５０％（２／４）。