Interleukin 12 and indomethacin exert a synergistic, angiogenesis-dependent antitumor activity in mice

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence and mortality from colorectal cancer. It has recently been demonstrated that these drugs are capable of suppressing the production of pro-angiogenic factors from tumor cells. The mechanisms of antitumor action of interleukin 12 include the enforced secretion of anti-angiogenic factors and stimulation of antitumor immunity. Therefore, we hypothesized that the combination of a model nonsteroidal anti-inflammatory drug--indomethacin and interleukin 12--would result in enhanced angiogenesis-dependent antitumor effects against a colon-26 carcinoma cells transplanted into syngeneic mice. As expected the combined administration of both agents simultaneously resulted in a strengthened antitumor activity that was manifested as a retardation of tumor growth and prolongation of mouse survival. Importantly some mice were completely cured after the combined treatment. As administration of interleukin 12 and indomethacin resulted in enhanced inhibition of angiogenesis it seems possible that prevention of new blood vessel formation is one of the mechanisms responsible for the observed antitumor effects.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Deposition of exotic bird-dispersed seeds into three habitats of a fragmented landscape in the northeastern United States

Dispersal by frugivorous birds facilitates invasion by many exotic plants. We measured the seed rain of ornithochorous native and exotic plants at three habitats of a fragmented landscape of the northeastern United States for 1 year. We studied maple-beech forests, old fields, and abandoned conifer plantations. Across all sites we collected 2,196 ornithochorous seeds, including seeds from six exotic species and 10 native species. The majority (90%) of collected seeds were from exotic species. Seed dispersal was broadly similar among habitats, though seed rain of exotic species was higher in old fields than forested habitats. Seed rain was not strongly influenced by artificial perches for most species. However, seeds of exotic species were more commonly found in traps under an artificial perch in old fields. Seed rains for the exotic Elaeagnus umbellata, Rhamnus cathartica, and Rosa multiflora were positively associated with local density of mature plants. Seed rain of R. cathartica was positively associated with abundance of seedlings but not saplings, suggesting that post-dispersal mortality was important. Seed dispersal of the exotic Lonicera spp. was high in all habitats, accounting for 66% of all seeds collected. With the exception of Lonicera spp., seed rain of common exotic invaders was affected by the abundance of seed sources, and these species might be effectively controlled by elimination of local fruiting plants. Fruits of Lonicera morrowii, which has extensively invaded our area, are apparently a common component in the diet of frugivores.     

An Evolution-Based Approach to De Novo Protein Design and Case Study on Mycobacterium tuberculosis

Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria Mycobacterium tuberculosis, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality.     

Epizootic emergence of Usutu virus in wild and captive birds in Germany

This study aimed to identify the causative agent of mass mortality in wild and captive birds in southwest Germany and to gather insights into the phylogenetic relationship and spatial distribution of the pathogen. Since June 2011, 223 dead birds were collected and tested for the presence of viral pathogens. Usutu virus (USUV) RNA was detected by real-time RT-PCR in 86 birds representing 6 species. The virus was isolated in cell culture from the heart of 18 Blackbirds (Turdus merula). USUV-specific antigen was demonstrated by immunohistochemistry in brain, heart, liver, and lung of infected Blackbirds. The complete polyprotein coding sequence was obtained by deep sequencing of liver and spleen samples of a dead Blackbird from Mannheim (BH65/11-02-03). Phylogenetic analysis of the German USUV strain BH65/11-02-03 revealed a close relationship with strain Vienna that caused mass mortality among birds in Austria in 2001. Wild birds from lowland river valleys in southwest Germany were mainly affected by USUV, but also birds kept in aviaries. Our data suggest that after the initial detection of USUV in German mosquitoes in 2010, the virus spread in 2011 and caused epizootics among wild and captive birds in southwest Germany. The data also indicate an increased risk of USUV infections in humans in Germany.     

Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.     

Effect of dietary fish oil on tumor-induced hyperlipidemia and tumor growth in rats implanted with a methylcholanthrene-induced sarcoma

Male Fischer 344 rats implanted with a methylcholanthrene-induced sarcoma (MCS), along with normal (or control) animals, were fed diets containing either 10% com oil (CO) or 2% CO + 8% fish oil (FO), designated as diets CO and FO, respectively, in a study designed to determine the effect of dietary FO on serum lipids (in the presence or absence of a tumor) and the growth and fatty acid composition of the MCS. For both diets, MCS-bearing rats had significantly (p < 0.05) higher serum levels of triglycerides, cholesterol, phospholipids, and total lipids than controls. For both controls and tumor-bearers, serum levels of all these lipids were, with the exception of cholesterol for the tumorbearers, significantly lower in rats receiving the FO diet than for the corresponding groups receiving the CO diet. Relative to rats fed the CO diet, those fed the FO diet had significantly higher serum levels of some fatty acids (e.g., 20:5n-3) but significantly lower levels of others (e.g., 18:2n-6), regardless of tumor status. For the tumor-bearers, differences in the levels of fatty acids in MCS tissue reflected differences in the fatty acid composition of total serum lipids. Sarcoma growth was unaffected by diet. Thus, feeding dietary FO resulted in changes in the lipid status of both control and tumor-bearing rats. Since sarcoma growth was unaffected by diet, the reduction in the severity of MCS-induced hyperlipidemia by FO appears to be due to an effect of the oil per se.