Advance Directives for Medical Decisions.

Patients can determine in advance how they want to be treated in a certain situation, and in particular, situations in which they reject treatment. The provisions to be observed for the preparation and practical implementation of a living will under German law are presented and discussed. The chapter also describes the principles according to which a decision is to be made if no living will has been drafted. Additionally, it is recommended that a trusted person should be granted power of attorney, since the future course of an illness (including cancer) cannot be predicted in every detail.

RAF inhibitor LY3009120 sensitizes RAS or BRAF mutant cancer to CDK4/6 inhibition by abemaciclib via superior inhibition of phospho-RB and suppression of cyclin D1.

KRAS, NRAS and BRAF mutations are among the most important oncogenic drivers in many major cancer types, such as melanoma, lung, colorectal and pancreatic cancer. There is currently no effective therapy for the treatment of RAS mutant cancers. LY3009120, a pan-RAF and RAF dimer inhibitor advanced to clinical study has been shown to inhibit both RAS and BRAF mutant cell proliferation in vitro and xenograft tumor growth in vivo. Abemaciclib, a CDK4/6-selective inhibitor, is currently in phase III studies for ER-positive breast cancer and KRAS mutant lung cancer. In this study, we found that combinatory treatment with LY3009120 and abemaciclib synergistically inhibited proliferation of tumor cells in vitro and led to tumor growth regression in xenograft models with a KRAS, NRAS or BRAF mutation at the doses of two drugs that were well tolerated in combination. Further in vitro screen in 328 tumor cell lines revealed that tumor cells with KRAS, NRAS or BRAF mutation, or cyclin D activation are more sensitive, whereas tumor cells with PTEN, PIK3CA, PIK3R1 or retinoblastoma (Rb) mutation are more resistant to this combination treatment. Molecular analysis revealed that abemaciclib alone inhibited Rb phosphorylation partially and caused an increase of cyclin D1. The combinatory treatment cooperatively demonstrated more complete inhibition of Rb phosphorylation, and LY3009120 suppressed the cyclin D1 upregulation mediated by abemaciclib. These results were further verified by CDK4/6 siRNA knockdown. Importantly, the more complete phospho-Rb inhibition and cyclin D1 suppression by LY3009120 and abemaciclib combination led to more significant cell cycle G0/G1 arrest of tumor cells. These preclinical findings suggest that combined inhibition of RAF and d-cyclin-dependent kinases might provide an effective approach to treat patients with tumors harboring mutations in RAS or RAF genes.

Dynamic Behavior of Trigger Factor on the Ribosome.

Trigger factor (TF) is the only ribosome-associated chaperone in bacteria. It interacts with hydrophobic segments in nascent chain (NCs) as they emerge from the ribosome. TF binds via its N-terminal ribosome-binding domain (RBD) mainly to ribosomal protein uL23 at the tunnel exit on the large ribosomal subunit. Whereas earlier structural data suggested that TF binds as a rigid molecule to the ribosome, recent comparisons of structural data on substrate-bound, ribosome-bound, and TF in solution from different species suggest that this chaperone is a rather flexible molecule. Here, we present two cryo-electron microscopy structures of TF bound to ribosomes translating an mRNA coding for a known TF substrate from Escherichia coli of a different length. The structures reveal distinct degrees of flexibility for the different TF domains, a conformational rearrangement of the RBD upon ribosome binding, and an increase in rigidity within TF when the NC is extended. Molecular dynamics simulations agree with these data and offer a molecular basis for these observations.

[New from old : relevant factors for fracture healing in aging bone]. / Aus alt mach neu : Relevante Faktoren bei der Frakturheilung im Alter.

BACKGROUND: Fracture healing is a complex biological process with specific temporal expression patterns. During this process new bone tissue is formed, which is similar to the original bone in quality and structure. This occurs in four phases: inflammation, formation of a soft tissue callus, formation of a bony callus and remodelling of the bony callus. This needs the precise orchestration of each cell type involved. OBJECTIVES: This article presents details of the fracture healing phases and the relevant factors. During the aging process there is an increase of reactive oxygen species and a change in expression pattern of growth factors that have a negative effect on the fracture healing process. METHODS: A selective review of the literature was carried out in PubMed concerning the influence of aging on fracture healing. CONCLUSION: The healing process is regulated by systemic and local factors. An understanding of these processes and the changes during aging is necessary in order to improve the knowledge of delayed or lack of fracture healing during aging to decide when an intervention is needed.

CHEK again: revisiting the development of CHK1 inhibitors for cancer therapy.

CHEK1 encodes the serine/threonine kinase CHK1, a central component of the DNA damage response. CHK1 regulates cell cycle checkpoints following genotoxic stress to prevent the entry of cells with damaged DNA into mitosis and coordinates various aspects of DNA repair. Accordingly, CHK1 has become a target of considerable interest in oncology. CHK1 inhibitors potentiate the efficacy of DNA-damaging chemotherapeutics by abrogating CHK1-mediated cell cycle arrest and preventing repair of damaged DNA. In addition, CHK1 inhibitors interfere with the biological role of CHK1 as a principal regulator of the cell cycle that controls the initiation of DNA replication, stabilizes replication forks, and coordinates mitosis. Since these functions of CHK1 facilitate progression through an unperturbed cell cycle, CHK1 inhibitors are being developed not only as chemopotentiators, but also as single-agent therapies. This review is intended to provide information on the current progress of CHK1 inhibitors in pre-clinical and clinical development and will focus on mechanisms of single-agent activity and potential strategies for patient tailoring and combinations with non-genotoxic agents.

[Rare retrovesical tumor manifestation of cystic echinococcosis (Echinococcus granulosus)]. / Seltene retrovesikale Tumormanifestation der zystischen Echinokokkose (Echinococcus granulosus).

Unclear pelvic retrovesical intraperitoneal tumors can be caused by cystic echinococcosis. A definitive diagnosis of this disease is highly problematic und often requires a qualified histological/parasitological assessment. It is not possible to diagnose or exclude a cystic echinococcosis purely on the basis of a serological diagnosis. A multiple organ infection can be excluded using CT diagnostics as described in this case. The Robert Koch Institute has to be notified in the case of a positive result.

PP015. Differential expression of Nrf2 and VEGF in human placental beds from normal and pregnancies complicated with preeclampsia and IUGR.

INTRODUCTION: Impaired trophoblast invasion into the uteroplacental arteries is accompanied with an evidence of oxidative stress in the extravillous trophoblast in preeclampsia complicated with IUGR. OBJECTIVES: Preeclampsia is characterised by increased lipid oxidation and diminished antioxidant capacity; recently, we have shown that PE is associated with an increased expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) in villous cytotrophoblast. A possible relationship between the vascular endothelial growth factor (VEGF) and Nrf2 was established in vitro and the activation of Nrf2 pathway could lead to upregulation of VEGF synthesis through the induction of Nrf2-dependent Heme oxygenase-1 (HO-1). In this study the expression of Nrf2 and VEGF was determined in the interstitial and intramural extravillous trophoblast in normal pregnancies and those complicated by preeclampsia and intra-uterine growth restriction (IUGR). METHODS: Full-thickness uterine tissues were obtained from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and from 5 women with severe early-onset preeclampsia and IUGR (29-34 week's gestation). The interstitial and intramural trophoblasts were studied by immunohistochemical analysis of paraffin sections stained with anti VEGF and anti Nrf2. RESULTS: Cases suffering from preeclampsia with IUGR were characterised by reduced invasion of extravillous trophoblast into uteroplacental arteries in the endometrial and myometrial segments. In addition, these cells showed an increased expression of Nrf2 in the pathological sections. The overexpression of Nrf2 in cases with preeclampsia was associated with restricted expression of VEGF in these cells compared to controls. CONCLUSION: Our data suggest that besides villous cytotrophoblast, also the extravillous trophoblast is a source of Nrf2-dependent genes. VEGF deficiency may cause higher oxidative stress in extravillous trophoblast in cases with preeclampsia with IUGR. The resulting reduced basal defence against oxidative stress and the higher vulnerability to oxidative damage may play a role in the limited trophoblast invasion into uteroplacental arteries in cases suffering from early onset preeclampsia and IUGR.

Transitory lupus anticoagulant antibodies leading to a quickly resolvable left ventricular thrombus in a young female patient on peritoneal dialysis.

We present a 38-year-old female patient on peritoneal dialysis for 3 years due to mesangioproliferative glomerulonephritis since early adolescence and chronic failure of the right kidney transplants. In early 2006 she was treated with high-dose cortisone due to cryptogenic, organized pneumonia. During a routine echocardiographic examination performed because of occurrence of cerebral symptoms such as diminished visual and auditory acuity in the patient, we detected a mobile, left ventricular thrombus of unusual large size, along with serologically measured Lupus anticoagulant antibodies (LA). The thrombus could be completely lyzed within only 12 hours by urokinese and antithrombotic danaparoid sodium therapy without surgical intervention. Successful treatment was proven by negative LA antibody activity as well as by echocardiography. The general clinical health was greatly improved after rehabilitation 2 months after lysis. We assume that the patient may have had infection- or cortisone-triggered transitory LA antibodies causing the serious heart thrombus with hypokinesia in the apex cordis.

Predictive value of plasma plasminogen activator inhibitor-1 for coronary restenosis: dependence on stent implantation and antithrombotic medication.

BACKGROUND: The plasmin activation system is involved in the development of restenosis after percutaneous coronary interventions (PCI). Conflicting data exist concerning the role of plasminogen activator inhibitor-1 (PAI-1) and its predictive value for restenosis. OBJECTIVES: To evaluate the fibrinolytic response to injury after PCI with or without stent implantation on different antithrombotic medications and its relation to late restenosis. PATIENTS AND METHODS: Eighty consecutive patients with successful PCI without (balloon only; n = 37) or with stent implantation (stent; n = 43) on different antithrombotic regimes (balloon only, aspirin; stent, aspirin/coumadin/dipyridamole vs. aspirin/ticlopidine). Blood samples were taken at baseline and up to 7 days after PCI and PAI-1 active antigen and tissue plasminogen activator (t-PA) antigen were determined. Restenosis was angiographically determined after 6 months. RESULTS: PCI increased both t-PA and PAI-1 levels (P < 0.001), with a significant prolonged and pronounced increase in stent vs. balloon-only patients (P < 0.05). Restenosis (stent 26%; balloon 38%) was significantly correlated to an attenuated PAI-1 increase after 24 h in the ticlopidine group (P = 0.007; restenosis, relative Delta PAI-1 + 50 +/- 28%; non-restenosis, + 139 +/- 50%), but not in the coumadin group. In the balloon-only group late restenosis (ISR) was associated with a trend for an augmented PAI-1 increase after 24 h. CONCLUSIONS: Coronary stent implantation significantly increases t-PA and PAI-1 plasma levels up to 1 week compared with balloon angioplasty alone. ISR in ticlopidine-treated patients was associated with an attenuated early PAI-1 active antigen increase. A less than 50% increase 24 h after stent implantation under ticlopidine treatment may identify patients at risk for the development of ISR.

Batch potency testing of inactivated erysipelas vaccines by ELISA--development, validation and implementation.

Inactivated erysipelas vaccines are widely used to protect pigs against erysipelas disease caused by the bacterium Erysipelothrix (E.) rhusiopathiae. Quality control tests for this vaccine are laid down in the European Pharmacopoeia (Ph.Eur.) Monograph No. 64. A laboratory animal model using a vaccination-challenge procedure is currently required as batch potency test. More than 10 years ago we initiated the first studies to develop an alternative ELISA potency model to replace this regulatory challenge test in mice. A short retrospective outline of the various steps from the development of the method until implementation into the regulatory requirements is described.

Architecture of the protein-conducting channel associated with the translating 80S ribosome.

In vitro assembled yeast ribosome-nascent chain complexes (RNCs) containing a signal sequence in the nascent chain were immunopurified and reconstituted with the purified protein-conducting channel (PCC) of yeast endoplasmic reticulum, the Sec61 complex. A cryo-EM reconstruction of the RNC-Sec61 complex at 15.4 A resolution shows a tRNA in the P site. Distinct rRNA elements and proteins of the large ribosomal subunit form four connections with the PCC across a gap of about 10-20 A. Binding of the PCC influences the position of the highly dynamic rRNA expansion segment 27. The RNC-bound Sec61 complex has a compact appearance and was estimated to be a trimer. We propose a binary model of cotranslational translocation entailing only two basic functional states of the translating ribosome-channel complex.

Structure of the 80S ribosome from Saccharomyces cerevisiae--tRNA-ribosome and subunit-subunit interactions.

A cryo-EM reconstruction of the translating yeast 80S ribosome was analyzed. Computationally separated rRNA and protein densities were used for docking of appropriately modified rRNA models and homology models of yeast ribosomal proteins. The core of the ribosome shows a remarkable degree of conservation. However, some significant differences in functionally important regions and dramatic changes in the periphery due to expansion segments and additional ribosomal proteins are evident. As in the bacterial ribosome, bridges between the subunits are mainly formed by RNA contacts. Four new bridges are present at the periphery. The position of the P site tRNA coincides precisely with its prokaryotic counterpart, with mainly rRNA contributing to its molecular environment. This analysis presents an exhaustive inventory of an eukaryotic ribosome at the molecular level.

Homocysteine plasma levels in young patients with coronary artery disease. Relation to history of acute myocardial infarction and anatomical extent of disease.

Although there is considerable epidemiologic evidence for a relationship between plasma homocysteine (Hcy) levels and cardiovascular disease, not all prospective studies have shown such a relationship. Furthermore, data concerning the role of hyperhomocysteinemia in patients with premature coronary artery disease (CAD) are rare. It was the aim of the study to investigate a possible association between Hcy plasma levels in young patients with the extent of CAD and the history of myocardial infarction (MI). A cohort of 94 patients was examined for conventional risk factors and the history of previous transmural MI. Furthermore, coronary angiography was performed to assess the anatomical extent of vessel disease. Plasma Hcy levels were measured by use of a commercial enzyme-linked immunosorbent assay. Only a history of previous MI was significantly associated with hyperhomocysteinemia. There was no relationship between elevated Hcy levels and the anatomical extent of vessel disease in patients with premature CAD. Our data may indicate that hyperhomocysteinemia represents an independent risk factor for acute coronary thrombosis rather than for the development of coronary sclerosis. Thereby, hyperhomocysteinemia may influence the clinical situation after plaque rupture not only by prothrombotic action but also by favouring endothelial dysfunction and vasospasm.

Impairment of the plasmin activation system in primary pulmonary hypertension: evidence for gender differences.

Primary pulmonary hypertension (PPH) is a rare disorder, with marked in-situ thrombosis of small pulmonary vessels occurring primarily in adult women. We investigated whether differences in the plasmin- and thrombin activation system are associated with the predominate affection of females. Plasma levels of plasminogen activator inhibitor type 1 (PAI-1), tissue-type plasminogen activator (t-PA), fibrinogen, thrombin-antithrombin (TAT) complexes, and prothrombin fragments (F1.2) were measured at baseline and after standardized venous occlusion (VO) in patients with PPH (24 female, 9 male). At baseline, females showed significant higher TAT levels (p = 0.05), higher t-PA antigen levels (p = 0.01) and higher fibrinogen levels (p = 0.03) with positive correlation to mean pulmonary artery pressure (mPAP), as well as nonsignificant lower t-PA activity, higher PAI-1 antigen and activity and F1.2 levels. After VO, females showed a significantly blunted increase in t-PA antigen (p = 0.01) and t-PA activity (p = 0.001), correlating with mPAP, as well as increased PAI-1 activity (p = 0.05). We hypothesize, that the observed presence of gender differences in the plasmin- and thrombin activation system in PPH leading to an antifibrinolytic/prothrombotic state might, in part, explain the female predominant incidence of this disease.

Stavudine versus zidovudine and the development of lipodystrophy.

The pathogenesis of some components of the lipodystrophy (LD) syndrome might be linked to the use of nucleosides. Earlier reports did not compare treatment regimens according to the nucleoside backbone. We studied a cohort of individuals who did not switch between stavudine and zidovudine. LD was defined to be present if one of three criteria was met: self-report by the patient, observation by an investigator who had known the patient since commencement of highly active antiretroviral therapy (HAART), or examination by a physician masked to therapy. The mean duration of therapy was 101 weeks (range: 26-234 weeks). Overall prevalence of LD was 48.7%. Lipoatrophy and lipohypertrophy occurred in 33.9% and 28.7% of patients, respectively. Logistic regression showed four parameters to be significantly associated with lipoatrophy: HAART longer than 2 years (p =.002, odds ratio [OR] = 4.4, 95% confidence interval [CI]: 1.608-11.965), baseline viral load >100,000 copies/ml (p =.004, OR = 4.3, CI: 1.726-11.197), age >40 years (p =.016, OR = 3.2, CI: 1.247-8.373), and white ethnicity (p =.041, OR = 5.4, CI: 1.070-28.184). Cholesterol levels of >200 mg/dl at baseline were associated with a risk reduction (p =.047, OR = 0.36, CI: 0.130-0.987). Use of lipohypertrophy as a dependent variable resulted in a significant association with HAART duration (p = 0.028, OR = 2.7, CI: 1.2-6.5) and protease inhibitor use (p =.014, OR = 3.8, CI: 1.3-11.2). LD prevalence is similar with both backbones using stavudine or zidovudine. This is the first time that baseline cholesterol was shown to be significantly associated with lipoatrophy.

Coexpression of band 3 mutants and Rh polypeptides: differential effects of band 3 on the expression of the Rh complex containing D polypeptide and the Rh complex containing CcEe polypeptide.

K562 cells were stably transfected with cDNAs encoding the band 3 found in Southeast Asian ovalocytosis (B3SAO, deletion of residues 400-408), band 3 with a transport-inactivating E681Q point mutation (B3EQ), or normal band 3 (B3). Flow cytometric analysis and quantitative immunoblotting revealed that B3SAO expressed alone was translocated to the plasma membrane, at levels similar to B3 or B3EQ. Nine monoclonal antibodies that reacted with extracellular loops of B3 also reacted with B3SAO, although the affinity of most antibodies for the mutant protein was reduced. Both known Wr(b) epitopes were expressed on K562/B3SAO cells, demonstrating that B3SAO interacts with glycophorin A. The growth rates of K562 clones expressing equivalent amounts of B3 and B3EQ were the same, suggesting that the potentially toxic transport function of band 3 may be regulated in K562 cells. The band 3-mediated enhancement of Rh antigen reactivity and the depression of Rh epitopes on SAO erythrocytes were investigated by comparing the coexpression of B3, B3SAO, or B3EQ in K562 clones expressing exogenous RhcE or RhD polypeptides. The results are consistent with an interaction between band 3 and the Rh polypeptide-Rh glycoprotein (RhAG) complex, which may enhance translocation of the complex or affect its conformation in the plasma membrane. The data suggest that the interaction between band 3 and the RhD-RhAG complex is weaker than it is between band 3 and the RhCcEe-RhAG complex.

Crystal structures of ribosome anti-association factor IF6.

Ribosome anti-association factor eIF6 (originally named according to translation initiation terminology as eukaryotic initiation factor 6) binds to the large ribosomal subunit, thereby preventing inappropriate interactions with the small subunit during initiation of protein synthesis. We have determined the X-ray structures of two IF6 homologs, Methanococcus jannaschii archaeal aIF6 and Sacchromyces cerevisiae eIF6, revealing a phylogenetically conserved 25 kDa protein consisting of five quasi identical alpha/beta subdomains arrayed about a five-fold axis of pseudosymmetry. Yeast eIF6 prevents ribosomal subunit association. Comparative protein structure modeling with other known archaeal and eukaryotic homologs demonstrated the presence of two conserved surface regions, one or both of which may bind the large ribosomal subunit.