Cognate peptide-receptor ligand mapping by directed phage display

BACKGROUND: A rapid phage display method for the elucidation of cognate peptide specific ligand for receptors is described. The approach may be readily integrated into the interface of genomic and proteomic studies to identify biologically relevant ligands. METHODS: A gene fragment library from influenza coat protein haemagglutinin (HA) gene was constructed by treating HA cDNA with DNAse I to create 50 – 100 bp fragments. These fragments were cloned into plasmid pORFES IV and in-frame inserts were selected. These in-frame fragment inserts were subsequently cloned into a filamentous phage display vector JC-M13-88 for surface display as fusions to a synthetic copy of gene VIII. Two well characterized antibodies, mAb 12CA5 and pAb 07431, directed against distinct known regions of HA were used to pan the library. RESULTS: Two linear epitopes, HA peptide 112 – 126 and 162–173, recognized by mAb 12CA5 and pAb 07431, respectively, were identified as the cognate epitopes. CONCLUSION: This approach is a useful alternative to conventional methods such as screening of overlapping synthetic peptide libraries or gene fragment expression libraries when searching for precise peptide protein interactions, and may be applied to functional proteomics

IgG_{4} Pf NPNA-1 a human anti-Plasmodium falciparum sporozoite monoclonal antibody cloned from a protected individual inhibits parasite invasion of hepatocytes

Malaria is one of the world's most devastating diseases, and Plasmodium falciparum (Pf) causes significant mortalities particularly in Sub-Saharan Africa. The rise and spread of multi-drug resistant strains of the parasite has coincided with an era of increased travel to malaria endemic regions. In the absence of an effective vaccine against malaria it may be possible to utilize human monoclonal antibodies against the stage transmitted by mosquito bites (sporozoites) as a prophylactic to prevent infection. We report the characterization of an engineered human IgG_{4} monoclonal antibody against Pf sporozoite cloned from a protected individual recognized the sporozoite surface and inhibited sporozoite invasion of human hepatocytes in vitro. The fully human monoclonal antibody PfNPNA-1 IgG_{4} against (NPNA)_{3} specifically labels Plasmodium falciparum in an IFA. This antibody also inhibits Plasmodium falciparum sporozoite invasion of human hepatocytes HepG2-A16 in a dose dependent manner in an in vitro assay. PfNPNA-1 IgG_{4} is a promising candidate for evaluation for the prevention of malaria

TMS320C6713DSP IMPLEMENTATION OF PULSE SHAPING FILTER

One of the most challenging issues facing deployment of 3G technology is how to make the network architectures compatible with each other. New signaling techniques are being designed specially to enhance today’s networks, deliver unprecedented functionality for 3G, and successfully derive the future generation of wireless systems, thus delivering immediate and long term benefits to subscribers. With the architecture of each generation of wireless devices addressed in the development of advance technologies, subscribers can easily evolve their systems without additional network modification, significantly reducing cost and implementing time. The next generation systems based on the DS-CDMA, FDMA/TDMA and GSM concepts are projected to provide transmitting high speed data, video and multimedia traffic for both indoor and outdoor systems, new technologies like Wideband Code Division Multiple Access (WCDMA), already in service, are providing users with high data rate services options like they have never experienced previously. The present paper deals with DSP Implementation of pulse shaping filter for wireless communication

Molecular dissection of the human antibody response to the structural repeat epitope of Plasmodium falciparum sporozoite from a protected donor

BACKGROUND: The circumsporozoite surface protein is the primary target of human antibodies against Plasmodium falciparum sporozoites, these antibodies are predominantly directed to the major repetitive epitope (Asn-Pro-Asn-Ala)(n), (NPNA)(n). In individuals immunized by the bites of irradiated Anopheles mosquitoes carrying P. falciparum sporozoites in their salivary glands, the anti-repeat response dominates and is thought by many to play a role in protective immunity. METHODS: The antibody repertoire from a protected individual immunized by the bites of irradiated P. falciparum infected Anopheles stephensi was recapitulated in a phage display library. Following affinity based selection against (NPNA)(3 )antibody fragments that recognized the PfCSP repeat epitope were rescued. RESULTS: Analysis of selected antibody fragments implied the response was restricted to a single antibody fragment consisting of V(H)3 and V(κ)I families for heavy and light chain respectively with moderate affinity for the ligand. CONCLUSION: The dissection of the protective antibody response against the repeat epitope revealed that the response was apparently restricted to a single V(H)/V(L )pairing (PfNPNA-1). The affinity for the ligand was in the μM range. If anti-repeat antibodies are involved in the protective immunity elicited by exposure to radiation attenuated P. falciparum sporozoites, then high circulating levels of antibodies against the repeat region may be more important than intrinsic high affinity for protection. The ability to attain and sustain high levels of anti-(NPNA)(n )will be one of the key determinants of efficacy for a vaccine that relies upon anti-PfCSP repeat antibodies as the primary mechanism of protective immunity against P. falciparum

Expression of recombinant multi-coloured fluorescent antibodies in gor -/trxB- E. coli cytoplasm

<p>Abstract</p> <p>Background</p> <p>Antibody-fluorophore conjugates are invaluable reagents used in contemporary molecular cell biology for imaging, cell sorting and tracking intracellular events. However they suffer in some cases from batch to batch variation, partial loss of binding and susceptibility to photo-bleaching. In theory, these issues can all be addressed by using recombinant antibody fused directly to genetically encoded fluorescent reporters. However, single-chain fragment variable domains linked by long flexible linkers are themselves prone to disassociation and aggregation, and in some cases with isoelectric points incompatible with use in physiologically relevant milieu. Here we describe a general approach that permits fully functional intracellular production of a range of coloured fluorescent recombinant antibodies with optimally orientated V_H/V_Linterfaces and isoelectric points compatible for use in physiological solutions at pH 7.4 with a binding site to fluorophore stoichiometry of 1:1.</p> <p>Results</p> <p>Here we report the design, assembly, intracellular bacterial production and purification of a panel of novel antibody fluorescent protein fusion constructs. The insertion of monomeric fluorescent protein derived from either <it>Discosoma </it>or <it>Aequorea </it>in-between the variable regions of anti-p185^HER2-ECDantibody 4D5-8 resulted in optimal V_H/V_Linterface interactions to create soluble coloured antibodies each with a single binding site, with isoelectric points of 6.5- 6. The fluorescent antibodies used in cell staining studies with SK-BR-3 cells retained the fluorophore properties and antibody specificity functions, whereas the conventional 4D5-8 single chain antibody with a (Gly₄Ser)₃linker precipitated at physiological pH 7.4.</p> <p>Conclusions</p> <p>This modular monomeric recombinant fluorescent antibody platform may be used to create a range of recombinant coloured antibody molecules for quantitative <it>in situ, in vivo </it>and <it>ex vivo </it>imaging, cell sorting and cell trafficking studies. Assembling the single chain antibody with monomeric fluorescent protein linker facilitates optimal variable domain pairing and alters the isoelectric point of the recombinant 4D5-8 protein conferring solubility at physiological pH 7.4. The efficient intracellular expression of these functional molecules opens up the possibility of developing an alternative approach for tagging intracellular targets with fluorescent proteins for a range of molecular cell biology imaging studies.</p

Investigations of the pi N total cross sections at high energies using new FESR: log nu or (log nu)^2

We propose to use rich informations on pi p total cross sections below N= 10 GeV in addition to high-energy data in order to discriminate whether these cross sections increase like log nu or (log nu)^2 at high energies, since it is difficult to discriminate between asymptotic log nu and (log nu)^2 fits from high-energy data alone. A finite-energy sum rule (FESR) which is derived in the spirit of the P' sum rule as well as the n=1 moment FESR have been required to constrain the high-energy parameters. We then searched for the best fit of pi p total cross sections above 70 GeV in terms of high-energy parameters constrained by these two FESR. We can show from this analysis that the (log nu)^2 behaviours is preferred to the log nu behaviours.Comment: to be published in Phys. Rev. D 5 pages, 2 eps figure

Neutrino Oscillations and Collider Test of the R-parity Violating Minimal Supergravity Model

We study the R-parity violating minimal supergravity models accounting for the observed neutrino masses and mixing, which can be tested in future collider experiments. The bi-large mixing can be explained by allowing five dominant tri-linear couplings $\lambda'_{1,2,3}$ and $\lambda_{1,2}$. The desired ratio of the atmospheric and solar neutrino mass-squared differences can be obtained in a very limited parameter space where the tree-level contribution is tuned to be suppressed. In this allowed region, we quantify the correlation between the three neutrino mixing angles and the tri-linear R-parity violating couplings. Qualitatively, the relations $| \lambda'_1 | < | \lambda'_2| \sim | \lambda'_3|$, and $|\lambda_1| \sim |\lambda_2|$ are required by the large atmospheric neutrino mixing angle $\theta_{23}$ and the small angle $\theta_{13}$, and the large solar neutrino mixing angle $\theta_{12}$, respectively. Such a prediction on the couplings can be tested in the next linear colliders by observing the branching ratios of the lightest supersymmetric particle (LSP). For the stau or the neutralino LSP, the ratio $|\lambda_1|^2: |\lambda_2|^2: |\lambda_1|^2 + |\lambda_2|^2$ can be measured by establishing $Br(e\nu): Br(\mu\nu) : Br(\tau\nu)$ or $Br(\nu e^\pm \tau^\mp ): Br(\nu\mu^\pm\tau^\mp) : Br(\nu\tau^\pm\tau^\mp)$, respectively. The information on the couplings $\lambda'_i$ can be drawn by measuring $Br(l_i t \bar{b}) \propto |\lambda'_i|^2$ if the neutralino LSP is heavier than the top quark.Comment: RevTex, 25 pages, 8 eps figure

Human monoclonal antibodies that neutralize anthrax toxin by inhibiting heptamer assembly

A panel of human anti-anthrax protective antigen IgG1 monoclonal antibodies were evaluated to determine the mechanism of toxin neutralization. AVP-22G12, AVP-1C6 and AVP-21D9 bound to the protective antigen with picomolar affinities to distinct non-overlapping linear epitopes. Two of the antibodies neutralized the anthrax toxin by completely inhibiting the protective antigen oligomer assembly process in vitro

Kang-Redner Anomaly in Cluster-Cluster Aggregation

The large time, small mass, asymptotic behavior of the average mass distribution \pb is studied in a $d$-dimensional system of diffusing aggregating particles for $1\leq d \leq 2$. By means of both a renormalization group computation as well as a direct re-summation of leading terms in the small reaction-rate expansion of the average mass distribution, it is shown that \pb \sim \frac{1}{t^d} (\frac{m^{1/d}}{\sqrt{t}})^{e_{KR}} for $m \ll t^{d/2}$, where $e_{KR}=\epsilon +O(\epsilon ^2)$ and $\epsilon =2-d$. In two dimensions, it is shown that \pb \sim \frac{\ln(m) \ln(t)}{t^2} for $m \ll t/ \ln(t)$. Numerical simulations in two dimensions supporting the analytical results are also presented.Comment: 11 pages, 6 figures, Revtex

Human anti-anthrax protective antigen neutralizing monoclonal antibodies derived from donors vaccinated with anthrax vaccine adsorbed

BACKGROUND: Potent anthrax toxin neutralizing human monoclonal antibodies were generated from peripheral blood lymphocytes obtained from Anthrax Vaccine Adsorbed (AVA) immune donors. The anti-anthrax toxin human monoclonal antibodies were evaluated for neutralization of anthrax lethal toxin in vivo in the Fisher 344 rat bolus toxin challenge model. METHODS: Human peripheral blood lymphocytes from AVA immunized donors were engrafted into severe combined immunodeficient (SCID) mice. Vaccination with anthrax protective antigen and lethal factor produced a significant increase in antigen specific human IgG in the mouse serum. The antibody producing lymphocytes were immortalized by hybridoma formation. The genes encoding the protective antibodies were rescued and stable cell lines expressing full-length human immunoglobulin were established. The antibodies were characterized by; (1) surface plasmon resonance; (2) inhibition of toxin in an in vitro mouse macrophage cell line protection assay and (3) in vivo in a Fischer 344 bolus lethal toxin challenge model. RESULTS: The range of antibodies generated were diverse with evidence of extensive hyper mutation, and all were of very high affinity for PA83~1 × 10(-10-11)M. Moreover all the antibodies were potent inhibitors of anthrax lethal toxin in vitro. A single IV dose of AVP-21D9 or AVP-22G12 was found to confer full protection with as little as 0.5× (AVP-21D9) and 1× (AVP-22G12) molar equivalence relative to the anthrax toxin in the rat challenge prophylaxis model. CONCLUSION: Here we describe a powerful technology to capture the recall antibody response to AVA vaccination and provide detailed molecular characterization of the protective human monoclonal antibodies. AVP-21D9, AVP-22G12 and AVP-1C6 protect rats from anthrax lethal toxin at low dose. Aglycosylated versions of the most potent antibodies are also protective in vivo, suggesting that lethal toxin neutralization is not Fc effector mediated. The protective effect of AVP-21D9 persists for at least one week in rats. These potent fully human anti-PA toxin-neutralizing antibodies are attractive candidates for prophylaxis and/or treatment against Anthrax Class A bioterrorism toxins