Antibody-independent activation of C1. I. Differences in the mechanism of C1 activation by nonimmune activators and by immune complexes: C1r-independent activation of C1s by cardiolipin vesicles

C1 activation is controlled by the regulatory protein C1-inhibitor (C1-INH). In contrast to immune-complex-induced activation, which is insensitive to C1-INH, antibody-independent activation of C1 is modulated by C1-INH. The mechanisms regulating nonimmune activation were studied with two phospholipids varying in their capacity to activate C1 in the presence of C1-INH: cardiolipin (CL) and phosphatidylglycerol (PG). Whereas C1-INH consistently suppressed activation by PG vesicles, a dose-dependent increase in C1 activation was measured with CL vesicles above 40 mole %. A similar dose-response binding of C1s requiring C1q, but not C1r, was detected only on CL vesicles, but neither on PG vesicles nor on immune complexes. This binding was Ca2+-dependent, suggesting that dimeric C1s is involved and was inhibited by spermine. The C1q-bound C1s was specifically cleaved at 37 degrees C into its active 58 kDa and 28 kDa chains, in the absence of C1r. On the addition of anti-CL antibodies, the C1q-mediated cleavage of C1s by CL vesicles was specifically inhibited. The cleavage of C1r on CL vesicles was also determined. When macromolecular C1 was offered in the presence of C1-INH, C1r cleavage was detected; however, the presence of C1s was a critical factor for C1r activation, because it was required on CL vesicles, but not on immune complexes. These results show that nonimmune activation of C1 presents specific features which distinguish it from immune complex-induced activation. These characteristics varied with the capacity of antibody-independent activators to activate C1 in the presence of C1-INH.     

Antibody-independent activation of C1, the first component of complement, by cardiolipin

Lipid vesicles containing phospholipids known to be present in substantial amounts in mitochondrial membranes were tested for their capacity to activate C1. Among them, only cardiolipin (CL) was highly efficient in C1 activation; no such effect was observed with phosphatidylcholine, phosphatidylethanolamine, or phosphatidylinositol. CL was shown to bind specifically C1q, because only unlabeled C1q competed with 125I-C1q for binding to CL. The requirement for C1q was confirmed by the finding that only fully reconstituted macromolecular C1, containing C1q, was activated by CL. The specificity of CL-induced activation of C1 was also demonstrated by introducing adriamycin, an agent known to interact with CL. Whereas adriamycin did not decrease C1 activation induced by immune complexes, it abrogated C1 activation by CL. The latter was shown to be a strong nonimmune activator of C1, because C1-INH did not inhibit CL-induced activation. When the concentration of CL in vesicles was decreased in the presence of phosphatidylcholine, C1 activation was detected only above a critical level of 35 mol% CL, compatible with a minimal density or clustering of CL molecules in the plane of the membrane. Moreover, C1 activation by CL was modulated by the addition of cholesterol. The threshold of CL required for C1 activation was lowered by the incorporation of more than 35 mol% cholesterol into the vesicles. These results show that CL incorporated into liposomes can be a potent nonimmune activator of C1. The negatively charged phosphate groups in CL are likely candidates for Clq-binding.     

Antibody-independent activation of C1. II. Evidence for two classes of nonimmune activators of the classical pathway of complement

Nonimmune activation of the first component of complement (C1) by cardiolipin (CL) vesicles present specific features which were not demonstrated on immune complexes. CL vesicles which activate C1 in the presence of C1-inhibitor (C1-INH) were found to bind C1s in the absence of C1r, and to induce a specific C1r-independent cleavage of C1q-bound C1s. Therefore, several known natural nonimmune activators were analyzed by comparing their ability to activate C1 in the presence of C1-INH and to mediate a C1r-independent cleavage of C1s. Freshly isolated human heart mitochondria (HHM) activated C1 only in the absence of C1-INH. However, mitoplasts derived from HHM (HHMP) activated C1 regardless of the presence of C1-INH, and induced a specific cleavage of C1q-bound C1s. The same pattern was observed in the case of smooth E. coli and a semi-rough E. coli strain. DNA, known to activate C1 only in the absence of C1-INH, does not induce C1s cleavage in the absence of C1r. Thus, nonimmune activators can be classified into two distinct categories. "Strong" activators, such as CL vesicles, HHMP, or the semi-rough E. coli strain J5 can activate C1 in the presence of C1-INH. By using C1qs2 as a probe, they exhibit a specific, C1r-independent cleavage of C1s. C1s-binding to C1q is a critical factor for the activation process in this group. In the case of "weak" activators, such as E. coli smooth strains, DNA, or HHM, no C1s-binding to activator-bound C1q was detected, and C1r-independent C1s cleavage and C1 activation in the presence of C1-INH were not observed. As in the case of immune complexes, C1r activation appears to play a key role in the C1 activation by "weak" activators.     

Sequence of E. coli tRNA-Glu1 by automated sequential degradation.

A minor tRNA-Glu1 constituent of a preparation of E. coli B tRNA-Glu (Oak Ridge) has a guanosine residue at position 66 rather than an adenosine as in the tRNA-Glu2 described by Ohashi et al. (3). Automated sequential degradation was used to sequence this region.     

PKC zeta participates in activation of inflammatory response induced by enteropathogenic E. coli

We showed previously that enteropathogenic Escherichia coli (EPEC) infection of intestinal epithelial cells induces inflammation by activating NF-B and upregulating IL-8 expression. We also reported that extracellular signal-regulated kinases (ERKs) participate in EPEC-induced NF-B activation but that other signaling molecules such as PKC may be involved. The aim of this study was to determine whether PKC is activated by EPEC and to investigate whether it also plays a role in EPEC-associated inflammation. EPEC infection induced the translocation of PKC from the cytosol to the membrane and its activation as determined by kinase activity assays. Inhibition of PKC by the pharmacological inhibitor rottlerin, the inhibitory myristoylated PKC pseudosubstrate (MYR-PKC-PS), or transient expression of a nonfunctional PKC significantly suppressed EPEC-induced IB phosphorylation. Although PKC can activate ERK, MYR-PKC-PS had no effect on EPEC-induced stimulation of this pathway, suggesting that they are independent events. PKC can regulate NF-B activation by interacting with and activating IB kinase (IKK). Coimmunoprecipitation studies showed that the association of PKC and IKK increased threefold 60 min after infection. Kinase activity assays using immunoprecipitated PKC-IKK complexes from infected intestinal epithelial cells and recombinant IB as a substrate showed a 2.5-fold increase in IB phosphorylation. PKC can also regulate NF-B by serine phosphorylation of the p65 subunit. Serine phosphorylation of p65 was increased after EPEC infection but could not be consistently attenuated by MYR-PKC-PS, suggesting that other signaling events may be involved in this particular arm of NF-B regulation. We speculate that EPEC infection of intestinal epithelial cells activates several signaling pathways including PKC and ERK that lead to NF-B activation, thus ensuring the proinflammatory response. inflammation; enteropathogenic Escherichia coli; nuclear factor-B; protein kinase C; IB kinase; extracellular signal-regulated kinase     

Antibody-independent interaction between the first component of human complement, C1, and the outer membrane of Escherichia coli D31 m4.

The heptoseless mutant of Escherichia coli, E. coli D31 m4, binds C1q and C1 at 0 degrees C and at low ionic strength (I0.07). Under these conditions, the maximum C1q binding averages 3.0 X 10(5) molecules per bacterium, with a Ka of 1.4 X 10(8) M-1. Binding involves the collagen-like region of C1q, as shown by the capacity of C1q pepsin-digest fragments to bind to E. coli D31 m4, and to compete with native C1q. Proenzyme and activated forms of C1 subcomponents C1r and C1s and their Ca2+-dependent association (C1r-C1s)2 do not bind to E. coli D31 m4. In contrast, the C1 complex binds very effectively, with an average fixation of 3.5 X 10(5) molecules per bacterium, and a Ka of 0.25 X 10(8) M-1, both comparable with the values obtained for C1q binding. C1 bound to E. coli D31 m4 undergoes rapid activation at 0 degrees C. The activation process is not affected by C1-inhibitor, and only slightly inhibited by p-nitrophenyl p'-guanidinobenzoate. No turnover of the (C1r-C1s)2 subunit is observed. Once activated, C1 is only partially dissociated by C1-inhibitor. Our observations are in favour of a strong association between C1 and the outer membrane of E. coli D31 m4, involving mainly the collagen-like moiety of C1.     

Adenovirus E3-early promoter: sequences required for activation by E1A.

To identify sequences within the adenovirus-5 E3 promoter necessary for E1A trans-activation, a series of promoter deletion mutants were constructed and analysed. A region between positions -82 and -105 was shown to be critical both for E1A induced expression as well as uninduced expression. The importance of this region was confirmed by constructing hybrid promoters consisting of E3 and Herpes simplex virus thymidine kinase sequences. The E1A insensitive tk promoter could be converted to an E1A sensitive promoter by replacing sequences upstream of position -79 with the corresponding region of the E3 promoter. This critical region of the E3 promoter contains a sequence 5' AGATGACTA3' which is also present in important upstream regions of the E2A and E4 promoters.     

Penicillin acylase production by E. coli

Enzyme production with E. coli ATCC 11105, in a complex medium using phenylacetic acid as inducer is carried out in a stirred-tank reactor of 10 dm³ and an airlift tower-loop reactor of 60 dm³ with outer loop at a temperature of 27 °C. The optimum inducer concentration was 0.8 kg/m³, which was kept constant by fed-batch operation. The optimum of the relative dissolved O₂-concentration with regard to saturation is below 10% in a stirred-tank reactor and at 35% in a tower-loop reactor. It was kept constant by parameter-adaptive control of the aeration rate. In a stirred-tank enzyme productivity is slightly higher than in a tower-loop reactor, and much higher than in a bubble column reactor.List of Symbols CPR kg/(m³ h) CO₂-production rate - OTR kg/(m³ h) O₂-transfer rate - OUR kg/(m³ h) O₂-utilization rate - PAA phenylacetic acid (inducer) - RQ = CPR/OUR respiratory quotient - X kg/m³ cell mass concentration - _m h^–1 maximum specific growth rate     

Separation of E. coli tRNA-Tyr precursor RNA from E. coli tRNA by hydroxyapatite chromatography

An assay procedure is described for triosephosphate isomerase based on measurement of the ellipticity of l-glyceraldehyde 3-phosphate remaining when d,l-glyceraldehyde 3-phosphate is the source of substrate and d-glyceraldehyde 3-phosphate is converted by triosephosphate isomerase to dihydroxyacetone phosphate. The assay method has advantages over the conventional coupled-enzyme assays in that it circumvents the difficulties posed by instability of the coupling enzymes and their cofactors, as well as by inhibitors of triosephosphate isomerase which may be present in preparations of the coupling enzymes. Although the method is not suited for routine assays during purification or in most clinical applications, it has advantages for detailed kinetic studies where pH, temperature, or other factors cause the coupled-enzyme assay procedures to be unreliable.     

肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中克隆的研究

在大肠杆菌中克隆肺炎支原体P1蛋白羧基端基因片段,为P1蛋白基因片段的扩增、表达及探讨羧基端基因片段功能打基础.采用PCR扩增方法获取P1结构基因.扩增产物用SalI和EcoRI酶切消化,回收1kb大小的DNA片段并与pUC19DNA连接,转入大肠杆菌JM109菌株.用X-gal平板及质粒图谱分析方法筛选重组克隆株,再用限制性核酸内切酶酶切图谱分析鉴定.经PCR扩增MPDNA获得1条5.0kbDNA片段.重组质粒限制性内切酶指纹图谱显示出2条带,1条为pUC19载体DNA带,另1条是1kb的插入片段.实验获得肺炎支原体P1蛋白结构基因及含P1蛋白羧基端DNA片段的重组克隆株.     

丙型肝炎病毒核心蛋白在大肠杆菌中的表达

目的：建立稳定表达丙型肝炎病毒(HCV)核心蛋白的原核表达系统,获得高产量的纯化核心蛋白。方法：应用多聚酶链反应(PCR),以HCV—H株全长cDNA序列为模板,扩增获得核心区基因片段,克隆入原核表达载体pBVIL1,构建原核表达载体pBVIL1-C,转化HB101宿主菌,通过温度诱导表达核心蛋白。结果：扩增得到目的基因长度为573bp,构建pBVIL1-C表达载体,在HB101宿主菌中通过温度诱导获得稳定表达,表达蛋白占菌体总蛋白含量的21％,Western—Blot检测证实表达产物可与HCV患者阳性血清发生特异性结合反应。结论：HCV核心蛋白可在大肠杆菌中获得高表达并具有良好的反应原性。