Crystal structures of the endoplasmic reticulum aminopeptidase-1 (ERAP1) reveal the molecular basis for N-terminal peptide trimming

Endoplasmatic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in trimming of peptides to an optimal length for presentation by major histocompatibility complex (MHC) class I molecules. Polymorphisms in ERAP1 have been associated with chronic inflammatory diseases, including ankylosing spondylitis (AS) and psoriasis, and subsequent in vitro enzyme studies suggest distinct catalytic properties of ERAP1 variants. To understand structure-activity relationships of this enzyme we determined crystal structures in open and closed states of human ERAP1, which provide the first snapshots along a catalytic path. ERAP1 is a zinc-metallopeptidase with typical H-E-X-X-H-(X)(18)-E zinc binding and G-A-M-E-N motifs characteristic for members of the gluzincin protease family. The structures reveal extensive domain movements, including an active site closure as well as three different open conformations, thus providing insights into the catalytic cycle. A K(528)R mutant strongly associated with AS in GWAS studies shows significantly altered peptide processing characteristics, which are possibly related to impaired interdomain interactions.     

Molecular evidence of transient therapeutic effectiveness of natalizumab despite high-titre neutralizing antibodies

Natalizumab is a humanized monoclonal antibody directed against the alpha-4 integrin subunit of very late activation antigen-4 (VLA-4). Natalizumab neutralizing antibodies (NAB) have been found to significantly reduce beneficial effects of natalizumab treatment in multiple sclerosis. We investigated interactions of NAB with natalizumab by serial measurements of alpha-4 integrin levels on peripheral blood mononuclear cells using flow cytometry. In addition, serum concentrations of soluble vascular cell adhesion molecule-1 (sVCAM-1), the endothelial ligand of VLA-4, and serum NAB were serially determined. Natalizumab infusion led to a transient reduction in alpha-4 integrin levels on immune cells and serum sVCAM-1 levels along with serum negativity of NAB lasting for a few days post-infusion. Apparently, the high-dose effect of freshly infused natalizumab resulted in a transient neutralization of NAB possibly involving a transient therapeutic effectiveness.     

The structure and protein kinase activity of proteins encoded by nonconditional mutants and back mutants in the sec gene of avian sarcoma virus

We have characterizedsrc proteins encoded by approximately 30 nonconditional transformation-defective mutants of avian sarcoma virus (ASV) and by several back mutants which reestablish a transformed phenotype. We used gel electrophoresis of immunoprecipitated proteins labeled with³²PO₄ or [³⁵S]methionine to assess size, stability, and phosphorylation; partial digestion with staphylococcal V8 protease to determine structure; and an immune complex assay to measure protein kinase activity. The mutants were all isolated as phenotypic revertants of the B31 line of B77-ASV transformed rat cells, each revertant cell bearing a single provirus without appreciable deletions, as described in the accompanying report (Varmuset al., 1980). In several instancesm the mutant proteins were examined both in the revertant rat cells and in chicken cells infected with transformation-defective viruses rescued from the nonpermissive rat cells. In addition, secondary mutations to restore a transformed phenotype (back mutations) occurred in some cases, in the original rat cells and/or chicken cells infected with rescued viruses. Three categories of mutants were identified by this survey. The largest group (Class I) encodedsrc proteins of normal size (60,000M_r); these proteins were hypophosphorylated and exhibited little or no protein kinase activity.Class II mutants displayed immunoprecipitablesrc proteins of less than normal size. In three cases, the shortsrc related proteins were mapped to the amino terminus of wild-type pp60^src and may be the result of nonsense mutations; in two cases, the short proteins were mapped to the car?yl terminus. Most of Class II mutants lacked protein kinase activity, but the 45,000M_r protein in line 000 exhibited moderate levels of activity, thereby mapping the enzymatically active site to the car?yl terminal three-fourths of pp60^src. The smallest group of mutants (Class III) did not produce detectablesrc proteins. Some of the mutant proteins behaved differently in permissive and nonpermissive hosts; in particular, the product of mutant L produced fusiform transformation and was highly phosphorylated and associated with wild-type levels of protein kinase activity in chicken cells, but was nontransforming, hypo-phosphorylated, and associated with low levels of protein kinase activity in rat cells. In all cases, back mutation to a transformed phenotype was accompanied by a restoration of wild-type (or near wild type) levels of protein kinase activity, further documenting the functional significance of the enzymatic activity. Some of the back mutants, however, encoded proteins of atypical size, either smaller or larger than pp60^src. The active proteins larger than pp60^src ranged up to 68,000M_r in size and were altered at or near the amino terminus. In one case (a retransformed derivative of the Class II revertant 000), the generation of a functionalsrc protein of 68,000M_r coincided with the appearance of an insert of ca. 200 base pairs into the ASV provirus, within or adjacent to the coding region for the amino terminus ofsrc. The diversity of reagents, both mutants and back mutants, derived from the single provirus in B31 cells indicates that this system will be useful for correlation of functional and structural attributes ofsrc.     

ACI大鼠肝细胞癌模型的免疫化疗栓塞术研究

目的:评价生物反应调节剂(BRM)经肝动脉化疗栓塞术(TACE)治疗ACI大鼠肝细胞癌的疗效。方法:在30只雄性ACI大鼠肝包膜下植入Morris Hepatom 3924A肝癌瘤块(2 mm3),移植术后13天行MRI检查,测量肿瘤体积V1;第14天时,经腹部切开术及胃十二指肠动脉逆行插管而采取以下3种介入治疗方案:①A组,0.1 mg丝裂霉素、0.1 ml碘油、7.5μg TNFα和5×103IU IL-2(n=10);②B组,0.1 mg丝裂霉素、0.1 ml碘油、0.05 mg链球菌提取物(OK-432)和5×103IU IL-2(n=10);③C组(对照组),0.1 mg丝裂霉素、0.1 ml碘油(n=10);13天后再次行MRI检查以确定肿瘤体积V2变化。比较肝肿瘤体积生长率V2/V1。结果:肿瘤治疗前和治疗后体积分别为A组0.0377 cm3和0.2752 cm3;B组0.0344 cm3和0.2233 cm3;C组0.0380 cm3和0.3398 cm3。介入治疗后肿瘤体积与治疗前肿瘤体积之比(V2/V1)分别为A组7.31、B组6.53、C组9.14。与对照组相比,采取A组的治疗方案能抑制肝肿瘤体积的生长(P=0.042),采取B组的治疗方案能明显抑制肿瘤的生长(P=0.004)。结论:采取免疫化疗栓塞术能有效抑制ACI大鼠肝细胞癌的生长。     

Verbessertes Überleben durch leitliniengerechte kardiopulmonale Reanimation

Background

In 2005 revised guidelines for cardiopulmonary resuscitation (CPR) were published by the European Resuscitation Council replacing the guidelines implemented in the year 2000. The aim of this study was to test the compliance with valid guidelines and to establish the quality of pre-hospital CPR provided by paramedics over a period of 38 months.

Patients and methods

A total of 299 CPRs performed by paramedics of the emergency medical services of Hamburg, Germany between 1^st November 2004 and 31^st December 2007 were analyzed. Digital recordings of automated external defibrillators and emergency protocol data were analyzed in detail. CPR was judged as incorrect if the defibrillation energy level did not correspond to the valid guidelines or if the interval between defibrillations exceeded a tolerance range of more than 30% compared to the valid guidelines.

Results

All CPRs (299) were included in the analysis of which 197 (65.9%) were intended to follow the 2000 guidelines and 102 (34.1%) the 2005 guidelines. Return of spontaneous circulation (ROSC) was achieved in 164 cases (54.8%) and survival to hospital admission in 125 cases (41.8%). CPR was performed accurately according to guidelines in only 26 cases (8.7%). In 273 cases (91.3%) the guidelines were not followed completely. Concerning the translation of guidelines into practice most faults occurred due to wrong intervals (89.3%), wrong defibrillation energy (33.4%) and medical errors, such as defibrillating an asystolic patient (7.0%). Primary survival rates were not significantly different when CPR accurately followed the 2000 or 2005 guidelines (40.1% versus 45.1%). Comparing primary survival rates of cases in which the guidelines were followed completely, there was no significant difference between the 2000 guidelines (15 out of 21 cases 71.4%) and 2005 guidelines (4 out of 5 cases 80.0%). However, compliance with valid guidelines significantly increased primary survival rates compared to non-compliance with valid guidelines (19 out of 26 cases 73.1% versus 106 out of 273 cases 38.8%; p=0.007). This effect was independent of the duration of CPR. Comparing CPR with monophasic defibrillation (189 cases) or biphasic defibrillation (58 cases), there was a significantly higher rate of ROSC (56.1% versus 72.4%) and a significantly higher rate of primary survival (41.3% versus 56.9%) in favour of biphasic defibrillation.

Conclusion

The results of our study show that compliance with valid guidelines is low and furthermore suggest that compliance with guidelines significantly reduces mortality. Future research may be warranted into the question of how to increase compliance with current CPR guidelines in pre-hospital emergency care.