New insights into porcine-human synteny conservation

Eleven genes were mapped to the porcine genome with the aim of improving the human-porcine comparative gene map. Five of these genes were from regions of the human genome painted by porcine chromosomal probes; of these, two mapped to chromosomes not expected from the painting results. Among the six genes from human regions not painted by porcine chromosomal probes, three genes did not map where expected by the principle of parsimony. Several of the gene assignments indicate the existence of small regions of conserved synteny not detected by heterologous chromosome painting, especially in telomeric regions. We have also detected new rearrangements in gene order within the regions of correspondence between human Chromosome (HSA) 15 and porcine Chromosome (SSC) 1 as well as between HSA4 and SSC8. Received: 30 September 1998 / Accepted: 3 December 1998     

Immunological comparison of glyoxalase I from yeast and mammals and quantitative determination of the enzyme in human tissues by radioimmunoassay

Antibodies to glyoxalase I from yeast, rat liver, porcine erythrocytes and human erythrocytes were raised in rabbits. Gel precipitation and immunotitration experiments demonstrated that the mammalian enzymes were immunologically related, but distinct from the yeast enzyme. Fab fragments of the antibodies to human glyoxalase I did not inhibit the catalytic activity, indicating that the antigen binding sites were not directed towards the active site of the enzyme. A radioimmunoassay for glyoxalase I was developed. Quantitative analysis of human adult as well as fetal organs demonstrated that glyoxalase I was present in a concentration of approximately 0.2 micrograms/mg protein in most human tissues.     

Molecular dynamics of Emiliania huxleyi and cooccurring viruses during two separate mesocosm studies

In this study we used denaturing gradient gel electrophoresis, sequencing analysis, and analytical flow cytometry to monitor the dynamics and genetic richness of Emiliania huxleyi isolates and cooccurring viruses during two mesocosm experiments in a Norwegian fjord in 2000 and 2003. We exploited variations in a gene encoding a protein with calcium-binding motifs (GPA) and in the major capsid protein (MCP) gene to assess allelic and genotypic richness within E. huxleyi and E. huxleyi-specific viruses (EhVs), respectively. To our knowledge, this is the first report that shows the effectiveness of the GPA gene for analysis of natural communities of E. huxleyi. Our results revealed the existence of a genetically rich, yet stable E. huxleyi and EhV community in the fjordic environment. Incredibly, the same virus and host genotypes dominated in separate studies conducted 3 years apart. Both E. huxleyi-dominated blooms contained the same six E. huxleyi alleles. In addition, despite the presence of at least six and four EhV genotypes at the start of the blooms in 2000 and 2003, respectively, the same two virus genotypes dominated the naturally occurring infections during the exponential and termination phases of the blooms in both years.     

Production of monocyte chemotactic and activating factor (MCAF) by human dermal fibroblasts in response to interleukin 1 or tumor necrosis factor

Normal human dermal fibroblasts rapidly expressed (less than 30 min.) considerable mRNA for monocyte chemotactic and activating factor (MCAF) and released high levels of biological activity in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF). In contrast, cultured normal human keratinocytes did not express MCAF mRNA when stimulated with IL 1 or TNF. These results suggest the important role of dermal fibroblasts, the predominant cells in dermal connective tissue, in the recruitment of monocytes during inflammation. This is the first report of the induction of MCAF by IL 1 or TNF in any cell type.     

Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping

The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces. Isolates of Bacillus subtilis (two isolates), Pseudomonas spp. (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D. flagrans growth on agar plates. When these isolates were added to cattle faeces containing D. flagrans and nematode larvae of C. oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D. flagrans. Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D. flagrans to trap and kill developing larvae of C. oncophora. Increasing chlamydospore doses of D. flagrans in faecal cultures resulted in higher nematode mortality. Thus, no evidence of interspecific or intraspecific competition was observed. The consequences of these findings are discussed.     

Seasonal and annual variability in the phytoplankton community of the Raunefjord,west coast of Norway from 2001–2006

Changes in phytoplankton community composition potentially affect the entire marine food web. Because of seasonal cycles and inter-annual variations in species composition, long-term monitoring, covering many sequential years, is required to establish a baseline study and to reveal long-term trends. The current study describes the phytoplankton biomass variations and species composition in relation to hydrographic and meteorological conditions in the Raunefjord, western Norway, over a 6-year period from 2001 to 2006. The extent of inflow or upwelling in the fjord varied from year to year and resulted in pronounced differences in water column stability. The annual phytoplankton community succession showed some repeated seasonal patterns, but also high variability between years. Two to four diatom blooms were observed per year, and the spring blooms occurring before water column stratification in March were dominated by Skeletonema marinoi and Chaetoceros socialis, and other Chaetoceros and Thalassiosira spp. Blooms of the haptophytes Phaeocystis pouchetii and Emiliania huxleyi were irregular and in some years totally absent. Although E. huxleyi was present all year round it appeared in bloom concentrations only in 2003, when the summer was warm and the water column characterized by high surface temperatures and pronounced stratification. The annual average abundance of both diatoms and flagellates increased during the six years. Despite the high variation from year to year, our investigation provides valuable knowledge about annual phytoplankton community patterns in the region, and can be used as a reference to detect possible future changes.     

Dynamics and reactivity of HbXL99 alpha. A cross-linked hemoglobin derivative

Resonance Raman spectroscopy, transient absorption, and fluroescence techniques have been employed to investigate the structure and dynamics of the alpha-cross-linked hemoglobin derivative, HbXL99 alpha. The resonance Raman spectra of the deoxy form of HbXL99 alpha are identical to those of native NbA (VFe-His approximately 222 cm-1), which exhibit a T-state (low affinity) structure regardless of solvent conditions. The resonance Raman spectra of the transient heme photoproduct resulting from CO photolysis from HbXL99 alpha appear to have structures intermediate between deoxy-T and ligand-bound R structures (VFe-His approximately 222 cm-1). Time-resolved resonance Raman data of HbXL99 alpha-CO show that complete CO recombination occurs after approximately 5 ms, with only a small amount of the CO-bound species reforming within approximately 200 ns (geminate recombination). Transient absorption spectra of HbXL99 alpha-O2 indicate that the extent of sub-nanosecond geminate recombination of O2 is also reduced in the cross-linked derivative relative to native HbA. The decrease in tryptophan fluorescence of HbXL99 alpha upon oxygenation further indicates that tertiary structural changes at the alpha 1-beta 2 interface upon ligation are apparently reduced, but not eliminated in the cross-linked derivative relative to HbA.     

NMR and interval PLS as reliable methods for determination of cholesterol in rodent lipoprotein fractions

Risk of cardiovascular disease is related to cholesterol distribution in different lipoprotein fractions. Lipoproteins in rodent model studies can only reliably be measured by time- and plasma-consuming fractionation. An alternative method to measure cholesterol distribution in the lipoprotein fractions in rat plasma is presented in this paper. Plasma from two rat studies (n = 68) was used in determining the lipoprotein profile by an established ultracentrifugation method and proton nuclear magnetic resonance (NMR) spectra of replicate samples was obtained. From the ultracentrifugation reference data and the NMR spectra, an interval partial least-square (iPLS) regression model to predict the amount of cholesterol in the different lipoprotein fractions was developed. The relative errors of the prediction models were between 12 and 33% and had correlation coefficients (r) between 0.96 and 0.84. The models were tested with an independent test set giving prediction errors between 19 and 46% and r between 0.96 and 0.76. Prediction of High, Low and Very Low Density Lipoprotein (HDL, LDL and VLDL) and total cholesterol was conducted in a study where rats had been supplemented with two doses of air-dried apple-powder. No significant difference in LDL, VLDL and total cholesterol was observed between the groups. The high apple-powder (20%) group had significantly lower HDL cholesterol (11%, P = 0.0452) than the control group. It is concluded that the iPLS approach yielded excellent regression models and thus univocal established chemometric analysis of NMR spectra of rat plasma as a strong and efficient way to quantify lipoprotein fractions in rat studies.     

Epitope grafting, re-creating a conformational Bet v 1 antibody epitope on the surface of the homologous apple allergen Mal d 1

Birch-allergic patients often experience oral allergy syndrome upon ingestion of vegetables and fruits, most prominently apple, that is caused by antibody cross-reactivity of the IgE antibodies in patients to proteins sharing molecular surface structures with the major birch pollen group 1 allergen from Betula verrucosa (Bet v 1). Still, to what extent two molecular surfaces need to be similar for clinically relevant antibody cross-reactivity to occur is unknown. Here, we describe the grafting of a defined conformational antibody epitope from Bet v 1 onto the surface of the homologous apple allergen Malus domestica group 1 (Mal d 1). Engineering of the epitope was accomplished by genetic engineering substituting amino acid residues in Mal d 1 differing between Bet v 1 and Mal d 1 within the epitope defined by the mAb BV16. The kinetic parameters characterizing the antibody binding interaction to Bet v 1 and to the mutated Mal d 1 variant, respectively, were assessed by Biacore experiments demonstrating indistinguishable binding kinetics. This demonstrates that a conformational epitope defined by a high affinity antibody-allergen interaction can successfully be grafted onto a homologous scaffold molecule without loss of epitope functionality. Furthermore, we show that increasing surface similarity to Bet v 1 of Mal d 1 variants by substitution of 6-8 residues increased the ability to trigger basophil histamine release with blood from birch-allergic patients not responding to natural Mal d 1. Conversely, reducing surface similarity to Bet v 1 of a Mal d 1 variant by substitution of three residues abolished histamine release in one patient reacting to Mal d 1.