Prediction of antigenic epitopes on protein surfaces by consensus scoring

Background  

Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance.     

The human T-cell lymphotropic virus type-I dimerization initiation site forms a hairpin loop, unlike previously characterized retroviral dimerization motifs

The formation of genomic RNA dimers during the retroviral life cycle is essential for optimal viral replication and infectivity. The sequences and RNA structures responsible for this interaction are located in the untranslated 5' leader RNA, along with other cis-acting signals. Dimer formation occurs by specific interaction between identical structural motifs. It is believed that an initial kissing hairpin forms following self-recognition by autocomplementary RNA loops, leading to formation of an extended stable duplex. The dimerization initiation site (DIS) of the deltaretrovirus human T-cell lymphotropic virus type-I (HTLV-I) has been previously localized to a 14-nucleotide sequence predicted to contain an RNA stem loop. Biochemical probing of the monomeric RNA structure using RNAse T1, RNAse V1, RNAse U2, lead acetate, and dimethyl sulfate has led to the generation of the first structural map of the HTLV-I DIS. A comprehensive data set of individual nucleotide modifications reveals that the structural motif responsible for HTLV-I RNA dimerization forms a trinucleotide RNA loop, unlike any previously characterized retroviral dimerization motif. Molecular modeling demonstrates that this can be formed by an unusual C:synG base pair closing the loop. Comparative phylogeny indicates that such a motif may also exist in other deltaretroviruses.     

Tapasin and other chaperones: models of the MHC class I loading complex

MHC (major histocompatibility complex) class I molecules bind intracellular virus-derived peptides in the endoplasmic reticulum (ER) and present them at the cell surface to cytotoxic T lymphocytes. Peptide-free class I molecules at the cell surface, however, could lead to aberrant T cell killing. Therefore, cells ensure that class I molecules bind high-affinity ligand peptides in the ER, and restrict the export of empty class I molecules to the Golgi apparatus. For both of these safeguard mechanisms, the MHC class I loading complex (which consists of the peptide transporter TAP, the chaperones tapasin and calreticulin, and the protein disulfide isomerase ERp57) plays a central role. This article reviews the actions of accessory proteins in the biogenesis of class I molecules, specifically the functions of the loading complex in high-affinity peptide binding and localization of class I molecules, and the known connections between these two regulatory mechanisms. It introduces new models for the mode of action of tapasin, the role of the class I loading complex in peptide editing, and the intracellular localization of class I molecules.     

Potassium permanganate as an in situ probe for B-Z and Z-Z junctions.

The availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.     

Mutationsversuche an Kulturpflanzen VI

Zusammenfassung der Ergebnisse Bei den mit angewandter Zielsetzung durchgeführten Mutationsversuchen an der Sojabohnensorte Heimkraft I wurden zunächst durch Triebkraftversuche Anhaltspunkte und dann im Freilandversuch genauere Hinweise für geeignete Röntgendosen für Bestrahlungsversuche mit Sojabohnen gefunden. Die Anzahl der Pflanzen mit Hülsenansatz der 6 kr-, 8 kr-, 10 kr-und 12 kr-Parzelle (35,0%, 15,3%, 21,8%, 15,5%) derX ₁-Generation zeigen, wie auch schon die im Gewächshaus durchgeführten Triebkraftversuche, daß im Gegensatz zu den AngabenGustafssons (1944) nach unseren Versuchen 10000 r nicht als Höchstmaß der Strahlenverträglichkeit von Sojabohnensamen angesehen werden kann. Im Triebkraftversuch waren bei einer Dosis von 16 kr nach fünf Wochen Versuchsdauer noch 12,5% der Pflanzen durchaus wüchsig, und erst bei 20 kr mit 0,7% wüchsigen Pflanzen war die letale Dosis nahezu erreicht.Wie die prozentuale Verteilung der insgesamt 427 bestätigten Mutanten auf die einzelnen Bestrahlungserien zeigt (Tab. II), sind Röntgendosen von 6 kr bis 12 kr, sowohl was die Höhe der Mutantenhäufigkeit als auch die Anzahl der überlebendenX ₁-Pflanzen (Tab. 4 und 5) betrifft, für Bestrahlungsversuche mit Sojabohnen am besten geeignet.Von den in unseren Versuchen gefundenen Mutanten haben nur einige reichverzweigte Formen, die frühreifen Typen, die Mutanten mit höherem Tausendkorngewicht und eine Reihe noch näher zu untersuchender Formen mit erhöhtem Hülsenbehang und Ertrag und geringerer Keimtemperatur züchterischen Wert. Die außer den Mutanten des Chlorophyllapparates noch zahlreich aufgetretenen verschiedenen Wuchstypen, die Veränderungen in der Blattform und Behaarung der Pflanzen und der Samenschalenfarbe, sind vom Standpunkt der deutschen Sojazüchtung als neutral oder in den meisten Fällen als negativ zu bezeichnen. Ihr Auftreten war aber insofern wichtig, als damit bewiesen werden kann, daß es auch bei Soja in verhältnismäßig kurzer Zeit möglich ist, aus einer Zuchtsorte ein Mutantensortiment experimentell zu erzeugen, in dem die charakteristischen Merkmale eines Teiles der im Weltsortiment bekannten Soja-Varietäten auftreten.Abgesehen davon, daß ein experimentell geschaffenes Mutantensortiment zur Lösung genetischer, physiologischer und biochemischer Fragestellungen geeignetes Ausgangsmaterial bietet, läßt sich aus den bisherigen Ergebnissen schließen, daß bei weiterer Arbeit in absehbarer Zeit Formen geschaffen werden können, die früher als die Ausgangssorte zur Reife kommen und ihr im Ertrag überlegen sind, Außerdem können die Mutanten mit züchterisch wertvollen Merkmalen als Ausgangsmaterial für weitere Kreuzungen verwendet werden und die schwierige Kombinationszüchtung der Sojabohne beschleunigen helfen.Mit 22 Textabbildungen     

ReFlexIn: A Flexible Receptor Protein-Ligand Docking Scheme Evaluated on HIV-1 Protease

For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation), that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1) protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.     

Intercellular chitinase and peroxidase activities associated with resistance conferred by geneLr35to leaf rust of wheat

The effect of leaf rust (Puccinia triticina) infection on intercellular chitinase (EC 3.2.1.14) and peroxidase (EC 1.11.1.7) activities was studied in resistant [RL 6082 (Thatcher/Lr35)] and susceptible (Thatcher) near isogenic wheat (Triticum aestivum L.) lines at seedling, stem elongation and flag leaf stages of plant growth. The levels of activity of these enzymes were low during the seedling and stem elongation stages. Resistant plants at the flag leaf stage, during which the Lr35 resistance gene was maximally expressed, exhibited high constitutive levels of chitinase and peroxidase activities, in contrast to the lower constitutive levels of susceptible plants. The results suggest that chitinase and peroxidase, constitutively present in the intercellular spaces of Thatcher/Lr35 wheat leaves, may play a role in Lr35 mediated resistance to leaf rust.