Basic and translational studies on species B adenoviruses

Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.

Protonation and deprotonation induced organo/hydrogelation: Bile acid derived gelators containing a basic side chain

Two bile acid derived molecules containing basic amino groups are reported to be efficient and unusual gelators of organic and aqueous solvents.

Reactions of the hexachlorocyclodiphosphazane [MeNPCl3]2 with primary aromatic amines: formation of highly basic bisphosphinimines

Reactions of hexachlorocyclodiphosphazane [MeNPCl3]2 with primary aromatic amines afforded the bisphosphinimine hydrochlorides [(RNH)2(RN)PN(Me)P(NHMe)(NHR)2]+Cl- (R = Ph 1, C6H4Me-4 2 or C6H4OMe-4 3). Dehydrochlorination of 2 and 3 by methanolic KOH yielded highly basic bisphosphinimines [(RNH)2(RN)PN(Me)P(NMe)(NHR)2] (R = C6H4Me-4 4 or C6H4OMe-4 5). Compounds 1-5 have been characterised by elemental analysis and IR and NMR (H-1, C-13, P-31) spectroscopy. The structure of 2 has been confirmed by single-crystal X-ray diffraction. The short P-N bond lengths and the conformations of the PN, units can be explained on the basis of cumulative negative hyperconjugative interactions between nitrogen lone pairs and adjacent P-N sigma* orbitals. Ab initio calculations on the model phosphinimine (H2N)3P=NH and its protonated form suggest that (amino)phosphinimines would be stronger bases compared to many organic bases such as guanidine.

Thermodynamics of lectin-sugar interaction: Binding of sugars to winged bean (Psophocarpus tetragonolobus) basic agglutinin (WBAI)

Combining site of WBAI is extended and encompasses all the residues of blood group A-reactive trisaccharide [GalNAcalpha3Galbeta4Glc]. Though both of the fucose residues of A-pentasaccharide [GalNAcalpha(Fucalpha2)3Galbeta(Fucalpha3)4Glc] do not directly interact, with the combining site they thermodynamically favour the interaction of GalNAcalpha3Galbeta4Glc part of the molecule by imposing a sterically favourable orientation of the binding epitope viz. GalNAcalpha3Galbeta4Glc of the saccharide. Binding of sugars is driven by enthalpy and is devoid of heat capacity changes. This together with enthalpy-entropy compensation observed for these processes underscore the importance of water reorganization as being one of the principal determinant of protein-sugar interactions.

Some Basic Aspects of Reaction Engineering of Precipitation Processes

Analysis of precipitation reactions is extremely important in the technology of production of fine particles from the liquid phase. The control of composition and particle size in precipitation processes requires careful analysis of the several reactions that comprise the precipitation system. Since precipitation systems involve several, rapid ionic dissociation reactions among other slower ones, the faster reactions may be assumed to be nearly at equilibrium. However, the elimination of species, and the consequent reduction of the system of equations, is an aspect of analysis fraught with the possibility of subtle errors related to the violation of conservation principles. This paper shows how such errors may be avoided systematically by relying on the methods of linear algebra. Applications are demonstrated by analyzing the reactions leading to the precipitation of calcium carbonate in a stirred tank reactor as well as in a single emulsion drop. Sample calculations show that supersaturation dynamics can assume forms that can lead to subsequent dissolution of particles that have once been precipitated.

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4'-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue. (C) Munksgaard 1995.

Structure of basic winged-bean lectin and a comparison with its saccharide-bound form

The crystal structure of the saccharide-free form of the basic form of winged-bean agglutinin (WBAI) has been solved by the molecular-replacement method and refined at 2.3 Angstrom resolution The final R factor is 19.74b for all data in the resolution range 8.0-2.3 Angstrom. The asymmetric unit contains two half-dimers, each located on a crystallographic twofold axis. The structure of the saccharide-free form is compared with that of the complex of WBAI wi th methyl-alpha-D-galactoside. The complex is composed of two dimers in the asymmetric unit. The intersubunit interactions in the dimer are nearly identical in the two structures The binding site of the saccharide-free structure contains three ordered water molecules at positions similar to those of the hydroxyl groups of the carbohydrate which an hydrogen bonded to the protein. Superposition of the saccharide-binding sites of the two structures shows that the major changes involve expulsion of these ordered water molecules and a shift of about 0.6 Angstrom of the main-chain atoms of the variable loop.

15-deoxyspergualin hinders physical interaction between basic residues of transit peptide in PfENR and Hsp70-1

The apicoplast of Plasmodium harbors several metabolic pathways. The enzymes required to perform these reactions are all nuclearly encoded and apicoplast targeted (NEAT) proteins. Plasmodium falciparum Enoyl-ACP Reductase (PfENR) is one such NEAT protein. The NEAT proteins have a transit peptide which is required for crossing the membranes of apicoplast. We studied the importance of basic residues like Arginine and Lysine within the transit peptide. Previous studies have suggested that all basic residues are essential for apicoplast trafficking. In this study, we demonstrate that only some of these residues are essential (K44, R48, K51, and R52), whereas others are dispensable (R40, K42, and K49). On mutating these specific residues, PfENR is not imported into the apicoplast and is mislocalized to the cytoplasm. We also demonstrate that these residues are also crucial for interaction with Hsp70-1, implying that interactions of Lysine 44, Arginine 48, Lysine 51, and Arginine 52 of the transit peptide with PfHsp70-1 are required for apicoplast trafficking. 15-Deoxyspergualin, which has earlier been proposed to interact with EEVD motif of PfHsp70-1 hinders the physical interaction between these cationic residues of PfENR and Hsp70-1. Hence, we propose that in the transport competent state of NEAT proteins some specific positively charged amino acids in the transit peptide interact with PfHsp70-1, and this interaction is essential for apicoplast targeting.

Basic principles of ultrafast Raman loss spectroscopy

When a light beam passes through any medium, the effects of interaction of light with the material depend on the field intensity. At low light intensities the response of materials remain linear to the amplitude of the applied electromagnetic field. But for sufficiently high intensities, the optical properties of materials are no longer linear to the amplitude of applied electromagnetic field. In such cases, the interaction of light waves with matter can result in the generation of new frequencies due to nonlinear processes such as higher harmonic generation and mixing of incident fields. One such nonlinear process, namely, the third order nonlinear spectroscopy has become a popular tool to study molecular structure. Thus, the spectroscopy based on the third order optical nonlinearity called stimulated Raman spectroscopy (SRS) is a tool to extract the structural and dynamical information about a molecular system. Ultrafast Raman loss spectroscopy (URLS) is analogous to SRS but is more sensitive than SRS. In this paper, we present the theoretical basis of SRS (URLS) techniques which have been developed in our laboratory.

Reactivity of Bispropargyl Sulfones under Basic Conditions: Interplay Between Garratt-Braverman and Schmittel/Myers-Saito Cyclization Pathway

The preference for GarrattBraverman (GB) over MyersSaito (MS) and Schmittel (SCM) cyclizations has recently been demonstrated in sulfones capable of undergoing all three of the processes. As the GB cyclization is a self-quenching process, there is a need to change the selectivity to the non-self-quenching MS or SCM pathway so as to enhance the DNA-cleaving efficiency that operates through the radical-mediated process. Herein we report a conformational constraint-based strategy developed by using computations (M06-2X/6-31+G*) to switch the selectivity from GB to MS/SCM pathway which also results in greater DNA-cleavage activity. The preference for GB could be brought back by easing the constraint with the help of spacers.