An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA

Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2.     

2,6-Disubstituted pyrazines and related analogs as NR2B site antagonists of the NMDA receptor with anti-depressant activity

Herein we describe the discovery of compounds that are competitive antagonists of the CP101-606 binding site within the NR2B subtype of the NMDA receptor. The compounds identified do not possess phenolic functional groups such as those in ifenprodil and related analogs. Initial identification of hits in this series focused on a basic, secondary amine side chain which led to good potency, but also presented a hERG liability. Further modifications led to examples of non-basic replacements which demonstrated much less liability in this regard. Finally, one compound in the series, 6a, was tested in the mouse forced swim depression assay and found to show activity (sc 60 mg/kg).     

An investigation into copper catalyzed D-penicillamine oxidation and subsequent hydrogen peroxide generation

D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.     

Centromeric histone H3 protein: from basic study to plant breeding applications

Centromere is the defining unit of a chromosome where kinetochore complex assembles and facilitates chromosome segregation. Centromeres contain unique repetitive sequences and are enriched with transposons and retrotransposons. Although how centromere is determined is still not clearly understood, binding of a key protein, namely, the Centromeric Histone H3 (CENH3) to centromeric repetitive DNA sequences has been found to be critical for the specification of centromere. Hence, centromeres are said to be epigenetically specified by CENH3. Despite considerable variation in size and sequence, CENH3 protein shows significant conservation of structure and function. CENH3 disruption or overexpression shows severe defects in spindle fiber attachment and ultimately leads to embryo lethality. Basic studies on complementation of CENH3 in Arabidopsis thaliana have led to the development of a novel method of haploid production through selective elimination of one set of parental chromosomes in the zygote. These findings have also shed new light on selective loss of chromosomes in interspecific crosses of Hordeum vulgare × H. bulbosum. Here, we briefly review unique features of CENH3 and discuss the new plant breeding opportunities that have emerged from the study of CENH3.     

An Evolutionarily Conserved Family of Virion Tail Needles Related to Bacteriophage P22 gp26: Correlation between Structural Stability and Length of the α-Helical Trimeric Coiled Coil

Bacteriophages of the Podoviridae family use short noncontractile tails to inject their genetic material into Gram-negative bacteria. In phage P22, the tail contains a thin needle, encoded by the phage gene 26, which is essential both for stabilization and for ejection of the packaged viral genome. Bioinformatic analysis of the N-terminal domain of gp26 (residues 1-60) led us to identify a family of genes encoding putative homologues of the tail needle gp26. To validate this idea experimentally and to explore their diversity, we cloned the gp26-like gene from phages HK620, Sf6 and HS1, and characterized these gene products in solution. All gp26-like factors contain an elongated α-helical coiled-coil core consisting of repeating, adjacent trimerization heptads and form trimeric fibers with length ranging between about 240 to 300 Å. gp26 tail needles display a high level of structural stability in solution, with T_m (temperature of melting) between 85 and 95 °C. To determine how the structural stability of these phage fibers correlates with the length of the α-helical core, we investigated the effect of insertions and deletions in the helical core. In the P22 tail needle, we identified an 85-residue-long helical domain, termed MiCRU (minimal coiled-coil repeat unit), that can be inserted in-frame inside the gp26 helical core, preserving the straight morphology of the fiber. Likewise, we were able to remove three quarters of the helical core of the HS1 tail needle, minimally decreasing the stability of the fiber. We conclude that in the gp26 family of tail needles, structural stability increases nonlinearly with the length of the α-helical core. Thus, the overall stability of these bacteriophage fibers is not solely dependent on the number of trimerization repeats in the α-helical core.     

Genetic diversity analysis of Leuconostoc mesenteroides from Korean vegetables and food products by multilocus sequence typing

Applied Microbiology and Biotechnology - In the present study, 35 Leuconostoc mesenteroides strains isolated from vegetables and food products from South Korea were studied by multilocus sequence...     

Design and development of novel N-(4-aminobenzoyl)- l-glutamic acid conjugated 1,3,5-triazine derivatives as Pf-DHFR inhibitor: An in-silico and in-vitro study

In the present work, a library of 120 compounds was prepared using various aliphatic and aromatic amines. Finally, 10 compounds were selected through in silico screening carrying 4-aminobenzoyl-l -glutamic acid and 1,3,5-triazine moiety. The docking results of compounds 4d16 and 4d38 revealed higher binding interaction with amino acids Asp54 (−537.96 kcal/mol) and Asp54, Phe116 (−618.22 kcal/mol) against wild (1J3I) and quadruple mutant (1J3K) type of Pf-DHFR inhibitors and were comparable to standard WR99210. These compounds were developed by facile and microwave-assisted synthesis via nucleophilic substitution reaction and characterized by different spectroscopic methods. In vitro antimalarial assay results also suggested that these two compounds were having higher antimalarial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strain out of the ten synthesized compounds with IC₅₀ 13.25 μM and 14.72 μM, respectively. These hybrid scaffolds might be useful in the lead discovery of a new class of Pf-DHFR inhibitors.