LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding.

Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS.WELLCOME TRUST; ward Number RG47206.This is the final version of the article. It first appeared from Elsevier at http://dx.doi.org/10.1016/j.pep.2010.11.018

Endothelial damage and dysfunction in acute graft-versus-host disease

Clinical studies suggested that endothelial dysfunction and damage could be involved in the development and severity of acute graft-versus-host disease (aGVHD). Accordingly, we found increased percentage of apoptotic Casp3+ blood vessels in duodenal and colonic mucosa biopsies of patients with severe aGVHD. In murine experimental aGVHD, we detected severe microstructural endothelial damage and reduced endothelial pericyte coverage accompanied by reduced expression of endothelial tight junction proteins leading to increased endothelial leakage in aGVHD target organs. During intestinal aGVHD, colonic vasculature structurally changed, reflected by increased vessel branching and vessel diameter. Because recent data demonstrated an association of endothelium-related factors and steroid refractory aGVHD (SR-aGVHD), we analyzed human biopsies and murine tissues from SR-aGVHD. We found extensive tissue damage but low levels of alloreactive T cell infiltration in target organs, providing the rationale for T-cell independent SR-aGVHD treatment strategies. Consequently, we tested the endothelium-protective PDE5 inhibitor sildenafil, which reduced apoptosis and improved metabolic activity of endothelial cells in vitro. Accordingly, sildenafil treatment improved survival and reduced target organ damage during experimental SR-aGVHD. Our results demonstrate extensive damage, structural changes, and dysfunction of the vasculature during aGVHD. Therapeutic intervention by endothelium-protecting agents is an attractive approach for SR-aGVHD complementing current anti-inflammatory treatment options

Comparative genome analysis of PHB gene family reveals deep evolutionary origins and diverse gene function

<p>Abstract</p> <p>Background</p> <p>PHB (Prohibitin) gene family is involved in a variety of functions important for different biological processes. PHB genes are ubiquitously present in divergent species from prokaryotes to eukaryotes. Human PHB genes have been found to be associated with various diseases. Recent studies by our group and others have shown diverse function of PHB genes in plants for development, senescence, defence, and others. Despite the importance of the PHB gene family, no comprehensive gene family analysis has been carried to evaluate the relatedness of PHB genes across different species. In order to better guide the gene function analysis and understand the evolution of the PHB gene family, we therefore carried out the comparative genome analysis of the PHB genes across different kingdoms.</p> <p>Results</p> <p>The relatedness, motif distribution, and intron/exon distribution all indicated that PHB genes is a relatively conserved gene family. The PHB genes can be classified into 5 classes and each class have a very deep evolutionary origin. The PHB genes within the class maintained the same motif patterns during the evolution. With<it> Arabidopsis</it> as the model species, we found that PHB gene intron/exon structure and domains are also conserved during the evolution. Despite being a conserved gene family, various gene duplication events led to the expansion of the PHB genes. Both segmental and tandem gene duplication were involved in Arabidopsis PHB gene family expansion. However, segmental duplication is predominant in Arabidopsis. Moreover, most of the duplicated genes experienced neofunctionalization. The results highlighted that PHB genes might be involved in important functions so that the duplicated genes are under the evolutionary pressure to derive new function.</p> <p>Conclusion</p> <p>PHB gene family is a conserved gene family and accounts for diverse but important biological functions based on the similar molecular mechanisms. The highly diverse biological function indicated that more research needs to be carried out to dissect the PHB gene function. The conserved gene evolution indicated that the study in the model species can be translated to human and mammalian studies.</p

Differentielle Immunisierungstechniken als eine Methode um neue Antigene zu entdecken, die mit Tumorprogression und Metastasierung assoziiert sind

I have continued characterizing a molecule that has already been identified in a previous subtractive immunization in order to get an idea of how this antigen works in normal tissue as well as in tumour cells. The M-N1 antigen seems to be very important for the progression of tumours and it plays a role in involuting breast tissue. The further elucidation of this function will certainly contribute to the comprehension of tumours. In the subtractive immunization I performed, I was able to isolate specific antibodies which recognize antigens on the highly metastasizing cell line AT6.1. These antigens also seem to play a role in tumour growth since the antibodies could clearly reduce tumour growth in experiments. Identifying and characterizing these antigens will give us further indications of necessary changes of the cell surface of tumour cells. There is still a blatant lack of molecules that can be identified in serum, which make possible a diagnosis of cancer at an early stage as well as to control the course of therapy. By the novel approach of serum immunization I tried to identify such molecules. I could isolate eight antibodies which in the serum discern molecules secreted or shedded by the tumour. One of these antibodies seemed to be particularly interesting since the antigen bound by it is expressed in many human tumour cell lines. I succeeded in identifying the antigens which turned out to be Prohibitin and BAP37. We do not yet known much about the functions of these two proteins. As a whole they are assigned a role in the mitochondria membrane as a chaperon to stabilize mitochondrial proteins. Prohibitin is supposed to interact with RB in the nucleus and thus to modulate the function of E2F. BAP37 under the name of REA (repressor of estrogen activity) is supposed to completively inhibit the ligation of co-activators to the estrogen receptor. Both proteins seem to play an important role in senescence. Also both proteins are apparently ligated to the IgM receptor in B-cells. Their function is unknown. Even though both proteins are found in the nucleus and in the cytoplasm, the antibody I isolated binds in immunofluorescent studies to prohibitin/BAP37 only in the cytoplasm. This may indicate a modified conformation possibly associated with a modified function. I am the first who could show a cell surface expression or a secretion for these proteins. (orig.)Bei der von mir durchgefuehrten subtraktiven Immunisierung konnte ich spezifische Antikoerper isolieren, die Antigene auf der hochmetastasierenden Zelllienie AT6.1 erkennen. Diese Antigene scheinen ebenfalls eine Funktion in der Tumorprogression zu spielen, da die Antikoerper in Versuchen das Tumorwachstum deutlich reduzieren konnten. Die Identifizierung und Charakterisierung dieser Antigene wird uns weitere Hinweise fuer die erforderlichen Veraenderungen von Tumorzellen auf ihrer Zelloberflaeche geben. Es besteht immer noch ein eklatanter Mangel an Molekuelen, die im Serum nachgewiesen werden koennen und die Diagnose von Krebs in fruehen Stadien ermoeglichen, aber auch den Verlauf einer Therapie ueberwachen koennen. In der von uns entwickelten Serum Immunisierung wurde von mir versucht, dem Ansatz, solche Molekuele zu identifizieren, gerecht zu werden. Ich konnte acht Antikoerper isolieren, die Molekuele im Serum erkennen, die vom Tumor sezerniert oder abgespalten werden. Einer dieser Antikoerper erschien als besonders interessant, da das von ihm gebundene Antigen auf vielen humanen Tumorzelllinien exprimiert wird. Es gelang mir, die Antigene zu identifizieren, die sich als Prohibitin und BAP37 herausstellten. Ueber die Funktionen beider Proteine ist noch nicht viel bekannt. Als Komplex wird ihnen eine Rolle in der Mitochondrienmembran als Chaperon zur Stabilisierung mitochondrialer Proteine zugesprochen. Prohibitin soll im Nukleus mit RB interagieren und somit die Funktion von E2F modulieren. BAP37 soll unter dem Namen REA (repressor of estrogen activity) das Binden von Koaktivatoren an den Oestrogenrezeptor kompetetiv inhibieren. Bei der Seneszenz scheinen beide Proteine eine wichtige Rolle zu haben. Auch sind beide Proteine anscheinend in B-Zellen an den IGM Rezeptor gebunden, welche Funktion sie dort haben, ist nicht bekannt. Obwohl beide Proteine im Nukleus und im Zytoplasma vorkommen, bindet mein isolierter Antikoerper in Immunfluoreszenzstudien nur im Zytoplasma, was vielleicht auf eine veraenderte Konformation schliessen laesst, die mit einer veraenderten Funktion einhergehen koennte. Ich konnte als erster fuer diese Proteine eine Zelloberflaechenexpression oder eine Sezernierung/Abspaltung zeigen. (orig.)Available from TIB Hannover: ZA 5141(6865) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Octane Index Applicability over the Pressure-Temperature Domain

Modern boosted spark-ignition (SI) engines and emerging advanced compression ignition (ACI) engines operate under conditions that deviate substantially from the conditions of conventional autoignition metrics, namely the research and motor octane numbers (RON and MON). The octane index (OI) is an emerging autoignition metric based on RON and MON which was developed to better describe fuel knock resistance over a broader range of engine conditions. Prior research at Oak Ridge National Laboratory (ORNL) identified that OI performs reasonably well under stoichiometric boosted conditions, but inconsistencies exist in the ability of OI to predict autoignition behavior under ACI strategies. Instead, the autoignition behavior under ACI operation was found to correlate more closely to fuel composition, suggesting fuel chemistry differences that are insensitive to the conditions of the RON and MON tests may become the dominant factor under these high efficiency operating conditions. This investigation builds on earlier work to study autoignition behavior over six pressure-temperature (PT) trajectories that correspond to a wide range of operating conditions, including boosted SI operation, partial fuel stratification (PFS), and spark-assisted compression ignition (SACI). A total of 12 different fuels were investigated, including the Co-Optima core fuels and five fuels that represent refinery-relevant blending streams. It was found that, for the ACI operating modes investigated here, the low temperature reactions dominate reactivity, similar to boosted SI operating conditions because their PT trajectories lay close to the RON trajectory. Additionally, the OI metric was found to adequately predict autoignition resistance over the PT domain, for the ACI conditions investigated here, and for fuels from different chemical families. This finding is in contrast with the prior study using a different type of ACI operation with different thermodynamic conditions, specifically a significantly higher temperature at the start of compression, illustrating that fuel response depends highly on the ACI strategy being used

Octane Index Applicability over the Pressure-Temperature Domain

Modern boosted spark-ignition (SI) engines and emerging advanced compression ignition (ACI) engines operate under conditions that deviate substantially from the conditions of conventional autoignition metrics, namely the research and motor octane numbers (RON and MON). The octane index (OI) is an emerging autoignition metric based on RON and MON which was developed to better describe fuel knock resistance over a broader range of engine conditions. Prior research at Oak Ridge National Laboratory (ORNL) identified that OI performs reasonably well under stoichiometric boosted conditions, but inconsistencies exist in the ability of OI to predict autoignition behavior under ACI strategies. Instead, the autoignition behavior under ACI operation was found to correlate more closely to fuel composition, suggesting fuel chemistry differences that are insensitive to the conditions of the RON and MON tests may become the dominant factor under these high efficiency operating conditions. This investigation builds on earlier work to study autoignition behavior over six pressure-temperature (PT) trajectories that correspond to a wide range of operating conditions, including boosted SI operation, partial fuel stratification (PFS), and spark-assisted compression ignition (SACI). A total of 12 different fuels were investigated, including the Co-Optima core fuels and five fuels that represent refinery-relevant blending streams. It was found that, for the ACI operating modes investigated here, the low temperature reactions dominate reactivity, similar to boosted SI operating conditions because their PT trajectories lay close to the RON trajectory. Additionally, the OI metric was found to adequately predict autoignition resistance over the PT domain, for the ACI conditions investigated here, and for fuels from different chemical families. This finding is in contrast with the prior study using a different type of ACI operation with different thermodynamic conditions, specifically a significantly higher temperature at the start of compression, illustrating that fuel response depends highly on the ACI strategy being used