Structure-activity relationships of the peptide deformylase inhibitor BB-3497: modification of the methylene spacer and the P1' side chain

Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial SAR around this lead for modifications to the methylene spacer and the P1' side chain. Enzyme inhibition and antibacterial activity data revealed that the optimum distance between the N-formyl hydroxylamine metal binding group and the P1' side chain is one unsubstituted methylene unit. Additionally, lipophilic P1' side chains that closely mimic the methionine residue in the substrate provided compounds with the best microbiological profile.     

Analysis of the bovine rumen microbiome reveals a diversity of Sus-like polysaccharide utilization loci from the bacterial phylum Bacteroidetes

Several unique Sus-like polysaccharide utilization loci (PULs) were identified from bacteria resident in bovine rumen microbiomes through functional screening of a fosmid library. The loci were phylogenetically assigned to the genus Prevotella within the phylum Bacteroidetes. These findings were augmented by a bioinformatic re-evaluation of ruminal Prevotella genomes, revealing additional loci not previously reported in the literature. Analysis of Bacteroidales-affiliated genomes reconstructed from a bovine rumen metagenome in a previous study further expanded the diversity of Sus-like PULs resident in this microbiome. Our findings suggest that Sus-like systems represent an important mechanism for degradation of a range of plant-derived glycans in ruminants.     

Structure and molecular mobility of soy glycinin in the solid state

We report a multitechnique study of structural organization and molecular mobility for soy glycinin at a low moisture content (<30% w/w) and relate these to its glass-to-rubber transition. Small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), Fourier transform infrared (FTIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy are used to probe structure and mobility on different length and time scales. NMR (approximately 10(-6) to 10(-3) s) reveals transitions at a higher moisture content (>17%) than DSC or SAXS, which sample for much longer times (approximately 10 to 10(3) s) and where changes are detected at >13% water content at 20 degrees C. The mobility transitions are accompanied by small changes in unit-cell parameters and IR band intensities and are associated with the enhanced motion of the polypeptide backbone. This study shows how characteristic features of the ordered regions of the protein (probed by SAXS and FTIR) and mobile segments (probed by NMR and DSC) can be separately monitored and integrated within a mobility transformation framework.     

Sporulation and Survival of Toxoplasma gondii Oocysts in Seawater

ABSTRACT. We have been collaborating since 1992 in studies on southern sea otters ( Enhdyra lutris nereis ) as part of a program to define factors, which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otiers. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study examined the sporulation of T. gondii oocysts in seawater and the survival of sporulated oocysts in seawater. Unsporulated oocysts were placed in 1.5 ppt artificial seawater, 32 ppt artificial seawater or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days post-inoculation under all treatment conditions. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatment groups. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in seawater. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt seawater at room temperature 22–24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate and remain viable in seawater for several months.     

Intra-Lesional Injection of the Novel PKC Activator EBC-46 Rapidly Ablates Tumors in Mouse Models

Intra-lesional chemotherapy for treatment of cutaneous malignancies has been used for many decades, allowing higher local drug concentrations and less toxicity than systemic agents. Here we describe a novel diterpene ester, EBC-46, and provide preclinical data supporting its use as an intra-lesional treatment. A single injection of EBC-46 caused rapid inflammation and influx of blood, followed by eschar formation and rapid tumor ablation in a range of syngeneic and xenograft models. EBC-46 induced oxidative burst from purified human polymorphonuclear cells, which was prevented by the Protein Kinase C inhibitor bisindolylmaleimide-1. EBC-46 activated a more specific subset of PKC isoforms (PKC-βI, -βII, -α and -γ) compared to the structurally related phorbol 12-myristate 13-acetate (PMA). Although EBC-46 showed threefold less potency for inhibiting cell growth than PMA in vitro, it was more effective for cure of tumors in vivo. No viable tumor cells were evident four hours after injection by ex vivo culture. Pharmacokinetic profiles from treated mice indicated that EBC-46 was retained preferentially within the tumor, and resulted in significantly greater local responses (erythema, oedema) following intra-lesional injection compared with injection into normal skin. The efficacy of EBC-46 was reduced by co-injection with bisindolylmaleimide-1. Loss of vascular integrity following treatment was demonstrated by an increased permeability of endothelial cell monolayers in vitro and by CD31 immunostaining of treated tumors in vivo. Our results demonstrate that a single intra-lesional injection of EBC-46 causes PKC-dependent hemorrhagic necrosis, rapid tumor cell death and ultimate cure of solid tumors in pre-clinical models of cancer.     

The Effect of Longitudinal Pre-Stretch and Radial Constraint on the Stress Distribution in the Vessel Wall: A New Hypothesis

It is well known that blood vessels shorten axially when excised. This is due to the perivascular tethering constraint by side branches and the existence of pre-stretch of blood vessels at the \textit {in situ} state. Furthermore, vessels are radially constrained to various extents by the surrounding tissues at physiological loading. Our hypothesis is that the axial pre-stretch and radial constraint by the surrounding tissue homogenizes the stress and strain distributions in the vessel wall. A finite element analysis of porcine coronary artery and rabbit thoracic aorta based on measured material properties, geometry, residual strain and physiological loading is used to compute the intramural stresses and strains. We systematically examined the effect of pre-stretch and external radial constraint in both vessels. Our results show that both stretching in the axial direction and compression in the radial direction lead to a more homogeneous strain and stress state in the blood vessel wall. A ``uniform biaxial strain' hypothesis is proposed for the blood vessel wall and the ramifications are discussed.     

High-Yield and Phylogenetically Robust Methods of DNA Recovery for Analysis of Microbial Biofilms Adherent to Plant Biomass in the Herbivore Gut

Recent studies have shown the microbial biofilms adherent to plant biomass in the gastrointestinal tracts of humans and other herbivores are quite different to planktonic populations. If these biofilm communities are to be properly characterized by metagenomics methods, then the microbial desorption methods used must ensure the phylogenetic diversity and genetic potential recovered is biologically valid. To that end, we describe here two different methods for desorbing microbes tightly adherent to plant biomass; and used PCR-DGGE analyses of the Bacteria and Archaea rrs genes to show both these desorption methods were effective in recovering the adherent microbial biofilm with no apparent biases in microbe recovery. We also present a derivation of the ??repeated bead beating and column (RBB+C) purification?? method of DNA extraction that results in the recovery of high molecular weight DNA. These DNA samples can be fragmented and size fractionated by sucrose density gradient centrifugation, bypassing the use of gel-plug lysis and pulsed-field gel electrophoresis separation of DNA for metagenomic library constructions.