Study on the interaction between 4-(1H-indol-3-yl)-2-(p-tolyl)quinazoline-3-oxide and human serum albumin

An organic small-molecular drug, 4-(1H-indol-3-yl)-2-(p-tolyl)quinazoline-3-oxide 1a was synthesized. It was employed to investigate the binding interaction and mechanism with human serum albumin (HSA). The experimental results indicated that the fluorescence quenching of HSA by 1a is a static quenching process and formation 1a-HSA complex. The site competition experiments revealed that the combination of 1a on HSA are hydrophobic interactions in the IIA domain and hydrogen bonds in IIIA domain of HSA, and the hydrophobic interactions of 1a on HSA are stronger than that of hydrogen bonds. These results were also confirmed by molecular docking theoretic analysis and ANS-hydrophobic fluorescent probe experiment. Synchronous fluorescence experiments showed that the polarity of HSA microenvironment was increase in the interaction process of 1a with HSA. The results of binding distance explored indicated that the combination distance between 1a and HSA is 3.63 nm, which is between 0.5R₀ and 1.5R₀, revealing the energy transfer between HSA and 1a is non-radiative. These results are very helpful for people to screen out high efficient indoloquinazoline drugs.     

Identification and synthesis of an efficient multivalent E. coli heat labile toxin inhibitor __ A dynamic combinatorial chemistry approach

A polymer based dynamic combinatorial library (DCL) was generated through condensation between aldehyde functionalized linear poly(glycidol) (APG) and galactose containing acylhydrazide derivatives. Pentameric E. coli heat labile enterotoxin B subunit (LTB) was subsequently applied to the DCL as external stimulus, resulting in amplification of a specific acylhydrazone side chain that was further used for the synthesis of a multivalent LTB inhibitor. In the in vitro biological evaluation, this inhibitor exhibited strong inhibition properties as well as low cytotoxicity.     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

Bimodal occurrence of aspartoacylase in myelin and cytosol of brain

The growing use of N-acetylaspartate as an indicator of neuronal viability has fostered interest in the biological function(s) of this unusual amino acid derivative. In considering the various physiological roles that have been proposed for this relatively abundant molecule one is obliged to take into account its unusual metabolic compartmentalization, according to which synthesis and storage occur in the neuron and hydrolytic cleavage in the oligodendrocyte. The latter reaction, catalyzed by aspartoacylase (ASPA), produces acetyl groups plus aspartate and has been proposed to occur in both soluble and membranous subfractions of white matter. Our study supports such bimodal occurrence and we now present immunoblot, proteomic, and biochemical evidence that the membrane-bound form of ASPA is intrinsic to purified myelin membranes. This was supported by a novel TLC-based method for the assay of ASPA. That observation, together with previous demonstrations of numerous lipid-synthesizing enzymes in myelin, suggests utilization of acetyl groups liberated by myelin-localized ASPA for lipid synthesis within the myelin sheath. Such synthesis might be selective and could explain the deficit of myelin lipids in animals lacking ASPA.     

The pivotal role of hepatocytes in drug discovery

This review promotes the value of isolated hepatocytes in modern Drug Discovery programmes and outlines how increased understanding, particularly in the area of in vitro-in vivo extrapolation (IVIVE), has led to more widespread use. The importance of in vitro metabolic intrinsic clearance data for predicting in vivo clearance has been acknowledged for several years and the greater utility of hepatocytes, compared with hepatic microsomes and liver slices, for this application is discussed. The application of hepatocytes in predicting drug-drug interactions (DDIs) resulting from reversible and irreversible (time-dependent) inhibition is relatively novel but affords the potential to study both phase I and phase II processes together with any impact of drug efflux and/or uptake (cellular accumulation). Progress in this area is reviewed along with current opinions on the comparative use of primary hepatocytes and higher throughput reporter gene-based systems for studying cytochrome P450 (CYP) induction. The appreciation of the role of transporter proteins in drug disposition continues to evolve. The study of hepatic uptake using isolated hepatocytes and the interplay between drug transport and metabolism with respect to both clearance and DDIs and subsequent IVIVE is also considered.     

QSAR of multiple mutated antibodies

The aim of this study was to develop predictive quantitative structure-activity relationship (QSAR) modeling for antibody-peptide interactions. A small single chain antibody library was designed and manufactured around the murine anti-p24 (HIV-1) monoclonal antibody CB4-1 by use of statistical molecular design (SMD) principles and site directed mutagenesis, and its affinity for a p24 derived antigen was determined by fluorescence polarization. A satisfactory QSAR model (Q(2) = 0.74, R(2) = 0.88) was derived by correlating the affinity data to physicochemical property scales of the amino acids varied in the library. The model explains most of the antibody-antigen interactions of the studied set, and provides insights into the molecular mechanism involved in antigen binding.     

A Century of B Chromosomes in Plants: So What?

BACKGROUND: Supernumerary B chromosomes (Bs) are a major source of intraspecific variation in nuclear DNA amounts in numerous species of plants. They favour large genomes, and create polymorphisms for DNA variation in natural populations. By studying Bs we can gain useful knowledge about the organization, function and evolution of genomes. There are also significant biological questions concerning the origin and structural organization of Bs, and the way in which these selfish elements can establish themselves by exploiting the replicative machinery of their host genome nucleus. SCOPE: It is a sine qua non that Bs originate from the A chromosomes, in a variety of ways. We can study their modes of drive and ask how it is that chromosomes which apparently lack genes can have control over their own drive process which leads to their survival in natural populations. Molecular cytogenetic studies are opening up new avenues of investigation. Population equilibria for B frequencies are determined by a balance between accumulation and harmful effects. Bs are also subject to meiotic loss due to polysomy and to elimination at meiosis as univalents. These balancing forces can be seen in the context of host/parasite interaction, based on a dissection of the genetic elements in both As and Bs (in maize) which interact to bring about a stable equilibrium, at least for a snapshot in time. CONCLUSIONS: Aside from their intrinsic enigmatic properties, B chromosomes make useful experimental tools to study genome organization. Thus far they have not been exploited for their applications, other than through the use of A-B translocations used for gene mapping in maize; but there are opportunities to use them to modulate the frequency and distribution of recombination, to diploidize allopolyploids, to study centromeres and to be developed as plant artificial chromosomes; given that they can be structurally modified and their inheritance stabilized.     

Pathogen fitness components and genotypes differ in their sensitivity to nutrient and temperature variation in a wild plant-pathogen association

Understanding processes maintaining variation in pathogen life-history stages affecting infectivity and reproduction is a key challenge in evolutionary ecology. Models of host-parasite coevolution are based on the assumption that genetic variation for host-parasite interactions is a significant cause of variation in infection, and that variation in environmental conditions does not overwhelm the genetic basis. However, surprisingly little is known about the stability of genotype-genotype interactions under variable environmental conditions. Here, using a naturally occurring plant-pathogen interaction, I tested whether the two distinct aspects of the infection process - infectivity and transmission potential - vary over realistic nutrient and temperature gradients. I show that the initial pathogen infectivity and host resistance responses are robust over the environmental gradients. However, for compatible responses there were striking differences in how different pathogen life-history stages and host and pathogen genotypes responded to environmental variation. For some pathogen genotypes even slight changes in temperature arrested spore production, rendering the developing infection ineffectual. The response of pathogen genotypes to environmental gradients varied in magnitude and even direction, so that their rankings changed across the abiotic gradients. Hence, the variable environment of spatially structured host-parasite interactions may strongly influence the maintenance of polymorphism in pathogen life-history stages governing transmission, whereas evolutionary trajectories of infectivity may be unaffected by the surrounding environment.     

Genetic quality of individuals impacts population dynamics

Ample evidence exists that an increase in the inbreeding level of a population reduces the value of fitness components such as fecundity and survival. It does not follow, however, that these decreases in the components of fitness impact population dynamics in a way that increases extinction risk, because virtually all species produce far more offspring than can actually survive. We analyzed the effects of the genetic quality (mean fitness) of individuals on the population growth rate of seven natural populations in each of two species of wolf spider in the genus Rabidosa , statistically controlling for environmental factors. We show that populations of different sizes, and different inbreeding levels, differ in population dynamics for both species. Differences in population growth rates are especially pronounced during stressful environmental conditions (low food availability) and the stressful environment affects smaller populations (<500 individuals) disproportionately. Thus, even in an invertebrate with an extremely high potential growth rate and strong density-dependent mortality rates, genetic factors contribute directly to population dynamics and, therefore, to extinction risk. This is only the second study to demonstrate an impact of the genetic quality of individual genotypes on population dynamics in a wild population and the first to document strong inbreeding–environment interactions for fitness among populations. Endangered species typically exist at sizes of a few hundred individuals and human activities degrade habitats making them innately more stressful (e.g. global climate change). Therefore, the interaction between genetic factors and environmental stress has important implications for efforts aimed at conserving the Earth's biodiversity.