Qualitative high field <Superscript>1</Superscript>H-NMR spectroscopy for the characterization of endogenous metabolites in earthworms with biochemical biomarker potential

This study was designed to provide a database of the endogenous metabolites in earthworm extracts of the species, Eisenia veneta and Lumbricus terrestris by high resolution ¹H-NMR spectroscopy in view of identifying biomarkers of toxicity or stress in environmental metabolomics studies. 1D and 2D NMR spectroscopic techniques enabled the identification and confirmation of the organic components in the tissue extracts of whole and segmented earthworms, dissected organs, and coelomic fluid. The extracts gave rise to characteristic ¹H-NMR spectral fingerprints of the low MW metabolites contained, specific to the species of earthworm, and to the specific regions or dissected organs of the earthworms under investigation. Distinct changes in the normal biochemistry were observed upon starvation and cooling, such as markedly decreased glucose and maltose, but increased lactate, acetate, succinate, formate and acetone. Additionally, slightly decreased threonine, arginine, lysine, leucine, citrate, asparagine and glycine were observed. Furthermore, lactate could be identified as a biomarker of acute toxic stress in expressed coelomic fluid following exposure to a model ecotoxin (3-trifluoromethylaniline). This work supports the application of ¹H-NMR spectroscopy for the study of changes in the normal invertebrate biochemistry in order to allow for the reliable assessment of biomarker responses following toxicity testing.     

Detection of chromosomal rearrangements derived from homologous recombination in four mapping populations of Brassica napus L

Genetic maps of Brassica napus were constructed from four segregating populations of doubled haploid lines. Each mapping population had the same male parent and used the same set of RFLP probes, facilitating the construction of a consensus map. Chromosomal rearrangements were identified in each population by molecular marker analysis and were classified as de novo homologous nonreciprocal transpositions (HNRTs), preexisting HNRTs, and homologous reciprocal transpositions (HRTs). Ninety-nine de novo HNRTs were identified by the presence of a few lines having duplication of a chromosomal region and loss of the corresponding homologous region. These de novo HNRTs were more prevalent in one population that had a resynthesized B. napus as a parent. Preexisting HNRTs were identified by fragment duplication or fragment loss in many DH lines due to the segregation of HNRTs preexisting in one of the parents. Nine preexisting HNRTs were identified in the three populations involving natural B. napus parents, which likely originated from previous homologous exchanges. The male parent had a previously described HRT between N7 and N16, which segregated in each population. These data suggest that chromosomal rearrangements caused by homologous recombination are widespread in B. napus. The effects of these rearrangements on allelic and phenotypic diversity are discussed.     

Structurally preserved sphenophytes from the Triassic of Antarctica: reproductive remains of Spaciinodum

Permineralized cones found organically attached to Spaciinodum collinsonii stems are described from the early Middle Triassic silicified flora from the Fremouw Formation of Antarctica, and the species diagnosis is emended to include the reproductive specimens. The apical cones are organized into internodal and leaf-bearing nodal regions. Nodal septations span the central pith and cortex, and thin fimbrils subdivide the internodal areas into smaller chambers. The vascular system consists of 31-33 continuous bundles that do not alternate in position between successive nodes and internodes. Simple sporangia are associated with the cortical chambers and occur in one whorl on the axis. Spores are small, lack elaters, and have no discernible ultrastructure preserved, and they are interpreted to be immature. The Antarctic cones are different in structure from typical cones of modern and fossil members of Equisetales; however, they share similarities with some morphologically aberrant cones of extant Equisetum and several Late Paleozoic and Mesozoic compression-impression fossils. Spaciinodum is now the most complete anatomically described Mesozoic sphenophyte.     

Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice

Purpose: Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA. Methods: The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN- from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 g/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10–14 and 17–21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days. Results: In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN- from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19–57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4⁺ and CD8⁺ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2. Conclusion: Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.     

A neural set point for the long-term control of arterial pressure: beyond the arterial baroreceptor reflex

Arterial baroreceptor reflex control of renal sympathetic nerve activity (RSNA) has been proposed to play a role in long-term control of arterial pressure. The hypothesis that the "set point" of the acute RSNA baroreflex curve determines the long-term level of arterial pressure is presented and challenged. Contrary to the hypothesis, studies on the long-term effects of sinoaortic denervation (SAD) on arterial pressure and RSNA, as well as more recent studies of chronic baroreceptor "unloading" on arterial pressure, suggest that the basal levels of sympathetic nerve activity and arterial pressure are regulated independent of arterial baroreceptor input to the brainstem. Studies of the effect of SAD on the long-term salt sensitivity of arterial pressure are consistent with a short-term role, rather than a long-term role for the arterial baroreceptor reflex in regulation of arterial pressure during changes in dietary salt intake. Renal denervation studies suggest that renal nerves contribute to maintenance of the basal levels of arterial pressure. However, evidence that baroreflex control of the kidney plays a role in the maintenance of arterial pressure during changes in dietary salt intake is lacking. It is proposed that a "baroreflex-independent" sympathetic control system must exist for the long-term regulation of sympathetic nerve activity and arterial pressure. The concept of a central nervous system "set point" for long-term control of mean arterial pressure (CNS-MAP set point), and its involvement in the pathogenesis of hypertension, is discussed.     

Determination and evaluation of an optimal dosage of carfentanil and xylazine for the immobilization of white-tailed deer (Odocoileus virginianus)

Using an iteration method, optimal hand-injected immobilization dosages of carfentanil/xylazine (CAR/XYL) were determined for 13 adult white-tailed deer (Odocoileus virginianus). Deer were temporarily restrained in a squeeze chute and were repeatedly immobilized one to four times at 2-5-wk intervals from December 2002 to March 2003. A fixed ratio of 1 mg CAR:10 mg XYL intramuscularly was used, increasing or decreasing the dosage until the optimal dosage (defined by an induction time < 3 min and PaCO(2)< 60 mmHg) was reached for each animal. Inductions were video-recorded and reviewed by observers blinded to drugs and dosages, who rated qualitative aspects of each induction. There were significant (P < 0.05) dosage-dependent decreases in induction time, time to first effect, PaO(2), SaO(2), and arterial pH, and significant dosage-dependent increases in PaCO(2) and quality ratings. The median optimal dosage (mOD) was 0.03 (range, 0.015-0.06) mg/kg CAR+0.3 (range, 0.15-0.6) mg/kg XYL. Induction times using the mOD were rapid (median 3.0 min [range, 1.8-10.0]), but quality ratings were considered undesirable for nine of 13 deer. Increased rectal body temperatures of 40.6+/-0.5 C (mean +/- SD) were noted in all deer and hyperthermia (T > 41 C) was noted in three. There was a positive correlation between body temperature and induction time (r=0.44). Heart rates significantly decreased from 5 to 15 min postinduction and remained decreased at the 20-min reading; there was occasional bradycardia. There was a significant increase in pH from 10 to 20 min postinduction, but metabolic acidemia (pH<7.3) persisted throughout the immobilization periods for all deer. Possible hypoxemia (SaO(2) and SpO(2)<90 mmHg but PaO(2)>60 mmHg) was present after induction, while hypercapnea (PaCO(2) > 60 mmHg) did not occur. Reversal times with naltrexone and yohimbine were rapid (mean 3.7+/-1.5 min) and uneventful, with no evidence of renarcotization. Although the median optimal dosage produced rapid inductions, no respiratory depression, complete reversal after antagonist administration, and no renarcotization, negative attributes included elevated body temperatures, acidemia, and undesirable induction qualities.     

Sensitivity of 70-mer oligonucleotides and cDNAs for microarray analysis of gene expression in Arabidopsis and its related species

Synthetic oligonucleotides (oligos) represent an attractive alternative to cDNA amplicons for spotted microarray analysis in a number of model organisms, including Arabidopsis, C. elegans, Drosophila, human, mouse and yeast. However, little is known about the relative effectiveness of 60-70-mer oligos and cDNAs for detecting gene expression changes. Using 192 pairs of Arabidopsis thaliana cDNAs and corresponding 70-mer oligos, we performed three sets of dye-swap experiments and used analysis of variance (anova) to compare sources of variation and sensitivities for detecting gene expression changes in A. thaliana, A. arenosa and Brassica oleracea. Our major findings were: (1) variation among different RNA preparations from the same tissue was small, but large variation among dye-labellings and slides indicates the need to replicate these factors; (2) sources of variation were similar for experiments with all three species, suggesting these feature types are effective for analysing gene expression in related species; (3) oligo and cDNA features had similar sensitivities for detecting expression changes and they identified a common subset of significant genes, but results from quantitative RT-PCR did not support the use of one over the other. These findings indicate that spotted oligos are at least as effective as cDNAs for microarray analyses of gene expression. We are using oligos designed from approximately 26,000 annotated genes of A. thaliana to study gene expression changes in Arabidopsis and Brassica polyploids.     

A locus for autosomal dominant "pure" hereditary spastic paraplegia maps to chromosome 19q13

Genetic loci for autosomal dominant pure hereditary spastic paraplegia (ADPHSP) have been mapped to chromosomes 2p, 8q, 12q, 14q, and 15q. We undertook a genomewide linkage screen of a large family with ADPHSP, for which linkage at all previously identified ADPHSP loci was excluded. Analysis of markers on chromosome 19q gave a peak pairwise LOD score of 3.72 at D19S420, allowing assignment of a novel ADPHSP locus (which we have termed "SPG12") to this region. Haplotype construction and analysis of recombination events narrowed the SPG12 locus to a 16.1-cM region between markers D19S868 and D19S902.