Eat now,pay later? Evidence of deferred food-processing costs in diving seals

Seals may delay costly physiological processes (e.g. digestion) that are incompatible with the physiological adjustments to diving until after periods of active foraging. We present unusual profiles of metabolic rate (MR) in grey seals measured during long-term simulation of foraging trips (4-5 days) that provide evidence for this. We measured extremely high MRs (up to almost seven times the baseline levels) and high heart rates during extended surface intervals, where the seals were motionless at the surface. These occurred most often during the night and occurred frequently many hours after the end of feeding bouts. The duration and amount of oxygen consumed above baseline levels during these events was correlated with the amount of food eaten, confirming that these metabolic peaks were related to the processing of food eaten during foraging periods earlier in the day. We suggest that these periods of high MR represent a payback of costs deferred during foraging.     

Proposed minimum reporting standards for chemical analysis

There is a general consensus that supports the need for standardized reporting of metadata or information describing large-scale metabolomics and other functional genomics data sets. Reporting of standard metadata provides a biological and empirical context for the data, facilitates experimental replication, and enables the re-interrogation and comparison of data by others. Accordingly, the Metabolomics Standards Initiative is building a general consensus concerning the minimum reporting standards for metabolomics experiments of which the Chemical Analysis Working Group (CAWG) is a member of this community effort. This article proposes the minimum reporting standards related to the chemical analysis aspects of metabolomics experiments including: sample preparation, experimental analysis, quality control, metabolite identification, and data pre-processing. These minimum standards currently focus mostly upon mass spectrometry and nuclear magnetic resonance spectroscopy due to the popularity of these techniques in metabolomics. However, additional input concerning other techniques is welcomed and can be provided via the CAWG on-line discussion forum at or . Further, community input related to this document can also be provided via this electronic forum. The contents of this paper do not necessarily reflect any position of the Government or the opinion of the Food and Drug Administration Sponsor: Metabolomics Society http://www.metabolomicssociety.org/ Reference: http://msi-workgroups.sourceforge.net/bio-metadata/reporting/pbc/ http://msi-workgroups.sourceforge.net/chemical-analysis/ Version: Revision: 5.1 Date: 09 January, 2007     

Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.     

Identification of distinct capsule types associated with Serratia marcescens infection isolates

Serratia marcescens is a versatile opportunistic pathogen that can cause a variety of infections, including bacteremia. Our previous work established that the capsule polysaccharide (CPS) biosynthesis and translocation locus contributes to the survival of S. marcescens in a murine model of bacteremia and in human serum. In this study, we determined the degree of capsule genetic diversity among S. marcescens isolates. Capsule loci (KL) were extracted from >300 S. marcescens genome sequences and compared. A phylogenetic comparison of KL sequences demonstrated a substantial level of KL diversity within S. marcescens as a species and a strong delineation between KL sequences originating from infection isolates versus environmental isolates. Strains from five of the identified KL types were selected for further study and electrophoretic analysis of purified CPS indicated the production of distinct glycans. Polysaccharide composition analysis confirmed this observation and identified the constituent monosaccharides for each strain. Two predominant infection-associated clades, designated KL1 and KL2, emerged from the capsule phylogeny. Bacteremia strains from KL1 and KL2 were determined to produce ketodeoxynonulonic acid and N-acetylneuraminic acid, two sialic acids that were not found in strains from other clades. Further investigation of KL1 and KL2 sequences identified two genes, designated neuA and neuB, that were hypothesized to encode sialic acid biosynthesis functions. Disruption of neuB in a KL1 isolate resulted in the loss of sialic acid and CPS production. The absence of sialic acid and CPS production also led to increased susceptibility to internalization by a human monocytic cell line, demonstrating that S. marcescens phagocytosis resistance requires CPS. Together, these results establish the capsule genetic repertoire of S. marcescens and identify infection-associated clades with sialic acid CPS components.     

Development of acetaminophen proline prodrug

In this research work, proline ester prodrug of acetaminophen (Pro-APAP) was synthesized and evaluated for its stability in PBS buffer at various pH and Caco-2 cell homogenate. The Pro-APAP is more stable at lower pH than higher pH, with half-life of 120 min in PBS buffer at pH 2.0, half-life of 65 min at pH 5.0, and half life of 3.5 min at pH 7.4, respectively. The half-life of Pro-APAP in Caco-2 cell homogenate is about 1 min, much shorter than the half-life in PBS buffer at pH 7.4, indicating enzymes in the cell homogenate contribute to the hydrolysis of the ester bond. Carboxypeptidase A was incubated with Pro-APAP at pH 7.4 with half-life of 3.8 min which is very close to the half life in buffer itself. This clearly indicates carboxypeptidase A is not one of the enzymes contributing to the hydrolysis of the prodrug. Physicochemical characteristics such as melting point and stability of newly synthesized prodrug were determined by MDSC technique.     

The role of endocytosis in regulating L1-mediated adhesion

L1 is a neural cell adhesion molecule critical for neural development. Full-length L1 (L1(FL)) contains an alternatively spliced cytoplasmic sequence, RSLE, which is absent in L1 expressed in nonneuronal cells. The RSLE sequence follows a tyrosine, creating an endocytic motif that allows rapid internalization via clathrin-mediated endocytosis. We hypothesized that L1(FL) would internalize more rapidly than L1 lacking the RSLE sequence (L1(Delta)(RSLE)) and that internalization might regulate L1-mediated adhesion. L1 internalization was measured by immunofluorescence microscopy and by uptake of (125)I-anti-rat-L1 antibody, demonstrating that L1(FL) is internalized 2-3 times faster than L1(Delta)(RSLE). Inhibition of clathrin-mediated endocytosis slowed internalization of L1(FL) but did not affect initial uptake of L1(Delta)(RSLE). To test whether L1 endocytosis regulates L1 adhesion, cell aggregation rates were tested. L1(Delta)(RSLE) cells aggregated two times faster than L1(FL) cells. Inhibition of clathrin-mediated endocytosis increases the aggregation rate of the L1(FL) cells to that of L1(Delta)(RSLE) cells. Our results demonstrate that rapid internalization of L1 dramatically affects L1 adhesion.     

Dissecting a charged network at the active site of orotidine-5'-phosphate decarboxylase

The crystal structure of yeast orotidine-5'-phosphate decarboxylase in complex with the postulated transition state analog, 6-hydroxyuridine-5'-phosphate, reveals contacts between this inhibitor and a novel quartet of charged residues (Lys-59, Asp-91, Lys-93, and Asp-96) within the active site. The structure also suggests a possible interaction between O2 of the 6-hydroxyuridine-5'-phosphate pyrimidine ring and Gln-215. Here we report the results of mutagenesis of each of the charged active site residues and Gln-215. The activities of the Q215A and wild-type enzymes were equal indicating that any interactions between this residue and the pyrimidine ring are dispensable for efficient decarboxylation. For the D91A and K93A mutant enzymes, activity was reduced by more than 5 orders of magnitude and substrate binding could not be detected by isothermal calorimetry. For the D96A mutant enzyme, k(cat) was reduced by more than 5 orders of magnitude, and isothermal calorimetry indicated an 11-fold decrease in the affinity of this enzyme for the substrate in the ground state. For the K59A enzyme, k(cat) was reduced by a factor of 130, and K(m) had increased by a factor of 900. These results indicate that the integrity of the network of charged residues is essential for transition state stabilization.     

Early intestinal Th1 inflammation and mucosal T cell recruitment during acute graft-versus-host reaction

Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells in the intestine during the induced graft-vs-host reaction (GVHR). We investigated the activation and mucosal homing phenotype of the donor CD4 cells and the kinetics of cytokine responses within the intestine and associated lymphoid tissues during early GVHR. Significant frequencies of donor CD4 cells accumulated within recipient Peyer's patches (PP), mesenteric lymph nodes (MLN), lamina propria (LP), and spleen (SP), during the first 9 days of GVHR. Many donor CD4 cells in SP, MLN, and LP expressed CD44 and also expressed de novo the mucosal homing integrin alpha(4)beta(7) (LPAM-1). A large IFN-gamma response occurred by day 3 in cells from PP and MLN, but much later (day 9) in SP and LP cells. IL-10 production by SP and MLN cells was elevated initially but declined substantially by day 9. IL-4 production by SP, MLN, and PP cells was low on day 3 and showed gradual decline in LP by day 9. IL-5 production by LP cells gradually increased in direct contrast to IL-5 production by MLN cells. The MLN CD4 cells showed the most dynamic changes, with high numbers of activated/effector donor CD4 cells and altered cytokine production consistent with a developing Th1 response. The IFN-gamma responses in PP and MLN preceded that of the SP, suggesting an intestinal origin for some Th1 effector cells in GVHR. Donor CD4 T cells apparently acquire the ability to home to the LP during early GVHR.     

Environmental contaminants and the prevalence of hemic neoplasia (leukemia) in the common mussel (Mytilus edulis complex) from Puget Sound, Washington, U.S.A

The relationship between hemic neoplasia, a blood cell disorder in bivalve molluscs, and chemical contaminants was evaluated in the common mussel (Mytilus edulis complex). Hemic neoplasia (HN) is endemic to mussel populations in Puget Sound. The prevalence of hemic neoplasia ranged from 0 to 30% in mussels from nine sites in Puget Sound, Washington. Organic chemical contamination in sediment from these sites range from 0.1 to 64.0 ppm of polycyclic aromatic hydrocarbons (PAHs) and 0.07 to 0.50 ppm chlorinated hydrocarbons. No relationship between the body burden of environmental contaminants and the prevalence of HN in mussels was identified. To evaluate the short-term ability of chemical contaminants to induce HN in mussels, mussels, from a site where mussels were previously determined to be HN free, were fed microencapsulated PAHs (composed of a mixture of phenanthrene, flouranthene, and benzo[a]pyrene) or PCBs (Aroclor 1254) and the prevalence of HN was assessed after 30 days of exposure. Although an apparent increase in HN prevalence (20 to 30%) was observed in all treatments groups except the untreated controls, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. A long-term (180-day) exposure study was conducted to evaluate the influence of PAHs or PCBs in modulating the prevalence of HN in a mussel population already exhibiting a moderate HN prevalence. Mussels, from a site where mussels were previously determined to exhibit a background prevalence of HN, fed microencapsulated PAHs, PCBs, and corn oil (vehicle) over a long time period (180 days), revealed an apparent increased prevalence of HN (30 to 40%) above the low levels (20%) initially present. However, no significant difference in the prevalence of HN was observed between the control group of mussels fed corn oil (vehicle) and mussels fed either PAHs or PCBs in corn oil. Although chemical contaminants have been proposed as a modulating factor in the development and promotion of HN in bivalve molluscs from environmentally stressed and degraded habitats, we find no evidence that chemical contaminants induce or promote the development of HN in the mussel M. edulis complex.