Evidence of impairment of normal inflammatory reaction by a high-fat diet.

The murine dorsal air pouch model is a valuable tool for studying acute peripheral inflammatory reactions. We used this model to study the effect of diet on the onset of acute inflammation. Mice were fed a normal or a high-fat diet (HFD) for 5 weeks. Air pouches were raised and injected with non-stimulating (saline) or stimulating solution (saline containing lipopolysaccharides). After 4 h, leukocytes in the pouch fluid were enumerated, sorted and their viability measured. Cytokine/chemokine levels in the cell-free fluid were measured using a cytometric bead assay. Gene expression level was measured in leukocytes and in lining tissues using comparative real-time PCR. Leukocyte migration and cytokine/chemokine secretion were decreased substantially in mice fed the HFD. In contrast, leptin levels were elevated. Gene expression profiles in leukocytes recovered from the pouch and in the pouch-lining tissue (believed to have an important role in the initiation of granulocyte recruitment) were depressed. Genes encoding CC and CXC family chemokines were among the most negatively affected. These results suggest that a HFD can alter peripheral tissue activation as well as leukocyte recruitment and response, thereby affecting the development of an effective local immune response, which could have deleterious consequences.

Increased tumorigenicity of cells carrying recombinant human herpesvirus 8.

Human herpesvirus 8 (HHV-8) infection is associated with the development of Kaposi's sarcoma and primary effusion lymphoma. The cloning of the HHV-8 genome into a bacterial artificial chromosome (BAC) allows researchers to mutate and identify the relative importance of HHV-8 genes essential for growth and replication in tissue culture systems. However, in vivo models to study the impact of such mutations are very limited. Consequently, the objective of this study was to determine whether cells carrying the HHV-8 BAC would form tumors when injected into mice, enabling the use of this model to assess the influence of viral gene mutation on tumorigenesis. To do so, 293T and 293T-E1 cells carrying recombinant HHV-8 were injected into SCID mice and tumor growth was analyzed. Our results clearly show that mice injected with 293T-E1 cells had a significantly higher tumor incidence level as well as increased tumor volumes and weights compared to mice injected with 293T control cells. Cells carrying the HHV-8 genome grew faster and more aggressively in SCID mice than control 293T cells, highlighting the oncogenic properties of HHV-8. The model presented could therefore be used for the identification of HHV-8 genes contributing to tumorigenesis in the context of the entire viral genome.

Improved large scale culture of Methylophilus methylotrophus for 13C/15N labeling and random fractional deuteration of ribonucleotides.

Isotopic labeling of RNA with 13C and 15N has become a routine procedure in structural studies by NMR spectroscopy. The methodology in this paper describes the random fractional deuteration of RNA using the obligate methylotropic bacterium, Methylophilus methylotrophus. This bacterium was grown using a non-deuterated carbon source in 52:48 D20/H20 and we have shown that all protons in the ribonucleotides except for the ribose H1 become 52% randomly fractionally deuterated. Improved growth conditions for this organism are also described that yield higher cell densities in liquid culture, which is applicable for all labeling procedures.

U- and Th-series radionuclides in snowshoe hare (Lepus americanus) taken near U mill tailings close to Elliot Lake, Ontario, Canada.

Snowshoe hare (Lepus americanus) trapped near U tailings had higher concentrations of (226)Ra in their bones (250 +/- 94 mBq g(-1) dry wt) than those from local control sites 3-15 km from the tailings (20-30 mBq g(-1) dry wt) and those from a distant control site 880 km away from the U mining area, which were below the detection limit (DL) (3.7 mBq g(-1) dry wt). Most chyme (stomach content) samples contained 226Ra below DL. Concentration ratios of 226Ra from tissues of local plants, considered important in the hare's diet, to bone ranged from 0.22 to 8.60. Concentrations of 210Pb and 210Po (95-245 mBq g(-1) dry wt) were not significantly different among tailings and control site populations. Disequilibrium between these isotopes and their precursors was noted. No significant accumulation of U and Th was noted at any site. Higher concentrations of 228Th compared to 232Th are attributed to accumulation of 228Ra in a manner similar to that of 226Ra. Based on bone 226Ra and 210Po contents, the maximum internal dose rates to the skeleton and the maximum life-time dose of hare living near tailings were 3.9 x 10(-5) Gy d(-1) and 4.2 x 10(-2) Gy, respectively. These rates were below the threshold required to produce osteosarcoma in other mammals and were considered unlikely to adversely affect hare during their lifetime. Radionuclide uptake by the animals was concluded to have no environmental significance in the transport of radionuclides from tailings to other locations.

Cytochrome P-450scc-mediated oxidation of (20S)-22-thiacholesterol: characterization of mechanism-based inhibition.

(20S)-22-thiacholesterol (1) is found to be a potent competitive inhibitor of pregnenolone biosynthesis from cholesterol by purified reconstituted bovine adrenal cytochrome P-450scc. The apparent dissociation constant Kd, determined from difference spectra, is 0.6 microM, close to the value from kinetic studies for the apparent inhibition constant, Ki, of 0.8 microM. Studies of the time course of pregnenolone production indicate that under turnover conditions the competitive inhibitor (1) is converted to a tighter binding inhibitor, shown to be (20S,22R)-22-thiacholesterol S-oxide (4), with high diastereoselectivity and in a time-dependent manner. Both the diastereomeric sulfoxides, (20S,22S)-22-thiacholesterol S-oxide (3) and (20S,22R)-22-thiacholesterol S-oxide (4), exhibit properties consistent with their being competitive versus cholesterol, but the (22R)-sulfoxide (4) binds approximately 10 times more tightly than the (22S) diastereomer (3). The apparent Kd values of sulfoxides 4 and 3 are 0.1 and 1.14 microM, respectively. EPR and absorption spectroscopic studies of enzyme-inhibitor complexes suggest direct coordination of the oxygen atom of the (22R)-sulfoxide (4) with the catalytic heme center. This implies that the inhibitor operates by directly blocking further reaction at the active site heme group, with a substantial lifetime of the enzyme-inhibitor complex.

Transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) genes in cell suspension cultures of Catharanthus roseus.

When cell suspension cultures of Catharanthus roseus are treated with autoclaved elicitor from the fungus Pythium aphanidermatum, they respond with the rapid transient induction of tryptophan decarboxylase (TDC) and strictosidine synthase (SS) enzyme activities, followed by the accumulation of indole alkaloids (Eilert et al., 1987). In this report, we demonstrate that expression of TDC and SS enzyme activities is preceeded by the transient appearance of mRNAs for both enzymes, suggesting transcriptional control of these events. The strong transient accumulation of both TDC and SS enzyme transcripts observed in elicitor-treated cell suspension cultures contrasts with the barely detectable level of TDC transcripts and the undetectable level of SS transcripts observed in developing seedlings.

Uptake of 226Ra by established vegetation and black cutworm larvae, Agrotis ipsilon (class Insecta: order Lepidoptera), on U mill tailings at Elliot Lake, Canada.

Radium-226 levels in samples from an inactive U tailings site at Elliot Lake, Ontario, Canada, were: 9,140 +/- 500 mBq g-1 dry weight in the substrate; 62 +/- 1 mBq g-1 dry weight in rye, Secale cereale, and less than 3.7 mBq g-1 dry weight in oats, Avena sativa, the dominant species established by revegetation of the tailings; and 117 +/- 7 mBq g-1 dry weight in washed and unwashed black cutworm larvae. Concentration ratios were: vegetation to tailings 0.001-0.007; black cutworms to vegetation 3.6 and black cutworms to tailings 0.01. The values are considered too low to be considered a hazard to herring gulls, Larus argentatus, which occasionally feed on cutworms.

Transfer coefficient of 226Ra from vegetation to meadow voles, Microtus pennsylvanicus, on U mill tailings.

The 226Ra level in vegetation growing on U mine tailings in Elliot Lake, Ontario, Canada, was 211 + 22 mBq g-1 (dry weight) compared to less than 7 mBq g-1 (dry weight) in material from a control site. Skeletons of meadow voles (Microtus pennsylvanicus) established on the tailings had concentrations of 226Ra of 6,083 +/- 673 mBq per animal in winter; 7,163 +/- 1,077 mBq per animal in spring; 1,506 +/- 625 mBq per animal in summer; and 703 +/- 59 mBq per animal in fall, compared to less than 7 mBq per animal in controls. The 226Ra transfer coefficient from vegetation to voles (defined as total millibecquerels of 226Ra in adult vole per total millibecquerels of 226Ra consumed by the vole in its lifetime) was calculated as 4.6 +/- 2.9 X 10(-2) in summer and 2.8 +/- 0.6 X 10(-2) in fall.