Noninvasive evaluation of liver metabolism by 2H and 13C NMR isotopomer analysis of human urine

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.     

Purification of antifreeze proteins by adsorption to ice

Antifreeze proteins (AFPs) can protect organisms from freezing injury by adsorbing to ice and inhibiting its growth. We describe here a method where ice, grown on a cold finger, is used to selectively adsorb and purify these ice-binding proteins from a crude mixture. Type III recombinant AFP was enriched approximately 50-fold after one round of partitioning into ice and purified to homogeneity by a second round. This method can also be used to purify non-ice-binding proteins by linkage to AFP domains as demonstrated by the recovery of a 50 kDa maltose-binding protein-AFP fusion from a crude lysate of Escherichia coli.     

NMR solution structure of attractin,a water-borne protein pheromone from the mollusk Aplysia californica

Water-borne protein pheromones are essential for coordination of reproductive activities in many marine organisms. In this paper, we describe the first structure of a pheromone protein from a marine organism, that of attractin (58 residues) from Aplysia californica. The NMR solution structure was determined from TOCSY, NOESY, and DQF-COSY measurements of recombinant attractin expressed in insect cells. The sequential resonance assignments were done with standard manual procedures. Approximately 90% of the 949 unambiguous NOESY cross-peaks were assigned automatically with simultaneous three-dimensional structure calculation using our NOAH/DIAMOD/FANTOM program suite. The final bundle of energy-refined structures is well-defined, with an average rmsd value to the mean structure of 0.72 +/- 0.12 A for backbone and 1.32 +/- 0.11 A for heavy atoms for amino acids 3-47. Attractin contains two antiparallel helices, made up of residues Ile9-Gln16 and I30-S36. The NMR distance constraints are consistent with the three disulfide bonds determined by mass spectroscopy (C4-C41, C13-C33, and C20-C26), where the first two could be directly determined from NOESY cross-peaks between CH beta protons of the corresponding cysteines. The second helix contains the (L/I)(29)IEECKTS(36) sequence conserved in attractins from five species of Aplysia that could interact with the receptor. The sequence and structure of this region are similar to those of the recognition helix of the Er-11 pheromone of the unicellular ciliate Euplotes raikovi, suggesting a possible common pathway for intercellular communication of these two distinct pheromone families.     

Improved islet survival and in vitro function using solubilized small intestinal submucosa

In vitro proliferation of isolated pancreaticislets has become an area of great interest given the scarcity of clinicalisletdonors and the islet mass requirements for clinical islet transplantation.Smallintestinal submucosa (SIS), a naturally occurring extracellular matrix, hasbeeninvestigated to promote wound healing, tissue remodeling and cell growth. Thisstudy evaluated recovery and function of isolated canine pancreatic isletsfollowing in vitro tissue culture. Pancreatic islets wereisolated from mongrel dogs using standard surgical procurement followed byintraductal collagenase distension, mechanical dissociation and EuroFicollpurification. Groups of purified islets were cultured in a humidifiedatmosphereof 95% air and 5% CO₂ for 48 hours in standard islet cultureconditions of CMRL 1066 tissue culture media (Gibco) which had beensupplementedwith 25M HEPES, penicillin/streptomycin and either 10% heat inactivatedfetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc.,West Lafayette, IN). The mean recovery of islets following the culture periodwas determined by sizing duplicate counts of a known volume and viability wasassessed by static incubation with low glucose (2.8 mM), highglucose (20 mM) and high glucose solution supplemented with 50m IBMX solution. Remaining islets were embeddedhistologically.From a consecutive series of six culture experiments, a significantly higher (p< 0.05) recovery of islets co-cultured with SIS was observed when comparedtocontrols. Mean islet recovery was 84.5 ± 2.9% (mean ± SEM) fromthe SIS cultured group compared with 64.7 ± 4.5% from the control groupcultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibiteda significantly higher (p <, 0.05) insulin response to the high glucosestimulus than islets cultured in the standard FCS cultured solution. Thecalculated stimulation index was 12.3 ± 3.4 for the SIS-treated groupcompared with 5.6 ± 1.8 for the standard cultured group (p < 0.05).The overall mean numbers of islets recovered following invitro culture was also higher in the SIS-treated group. Theproportion of islets with a mean diameter >150 m increasedfrom 24% to 31% in the SIS-treated group, whereas the same proportion decreasedto 18% from 22% in the control (FCS-treated) group. Histological evaluation offixed tissue samples collected following the culture period identified insulinand glucagon-secreting cells in the SIS and FCS treated groups, however ahigherfrequency of insulin positive cells were detected consistently in the SIStreated group. A proliferation marker (PCNA) identified positive cells withinboth groups as well. This study suggests that co-culture of freshly isolatedcanine islets in medium supplemented with solubilized SIS can improve thepost-culture recovery and in vitro islet function. Futureinvestigations will focus on the cellular interactions of SIS, bothinvitro and in vivo.     

Foraging efficiency of juvenile bluegill, Lepomis macrochirus, among different vegetated habitats

Optimal foraging theory is devoted to understanding how organisms maximize net energy gain. However, both the theory and empirical studies lack critical components, such as effects of environmental variables across habitats. We addressed the hypothesis that energetic returns of juvenile bluegill are affected by environmental variables characteristic of the vegetated habitats. Predicted optimal diet breadths were calculated and compared to prey items eaten by juvenile bluegill to determine if bluegill were foraging to maximize energetic gain. Differences in habitat profitability among vegetated sites were determined by comparing predictions of maximized energetic return rates (cals^-1) with prey contents of bluegill stomachs. Sizes of most prey items eaten by juvenile bluegill throughout the vegetated sites were smaller than the predicted optimal diet breadths. However, inclusion of smaller prey items in the diet did not seem to affect rate of energetic gain. Energetic return rates were maximized at the 1.5 and 2mm prey size classes and declined only slightly with inclusion of smaller prey sizes. Predicted energetic return rates and average mass in bluegill stomachs were related negatively. Average mass in bluegill stomachs also was associated negatively with Elodea canadensis stem densities and percent of light transfer, suggesting that foraging efficiency of bluegill decreased as plant density and percent of light increased. Results of our research indicate that maximization of energetic return rates is dependent upon availability of prey sizes that contribute to optimal foraging. Thus, determination of those habitats that provide the highest availability of benthic invertebrate prey with the least interference by stems is critical. Enhanced foraging capabilities can promote recruitment, faster growth, better body condition and survival.     

Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.     

Microbial composition of near-boiling silica-depositing thermal springs throughout Yellowstone National Park

The extent of hyperthermophilic microbial diversity associated with siliceous sinter (geyserite) was characterized in seven near-boiling silica-depositing springs throughout Yellowstone National Park using environmental PCR amplification of small-subunit rRNA genes (SSU rDNA), large-subunit rDNA, and the internal transcribed spacer (ITS). We found that Thermocrinis ruber, a member of the order Aquificales, is ubiquitous, an indication that primary production in these springs is driven by hydrogen oxidation. Several other lineages with no known close relatives were identified that branch among the hyperthermophilic bacteria. Although they all branch deep in the bacterial tree, the precise phylogenetic placement of many of these lineages is unresolved at this time. While some springs contained a fair amount of phylogenetic diversity, others did not. Within the same spring, communities in the subaqueous environment were not appreciably different than those in the splash zone at the edge of the pool, although a greater number of phylotypes was found along the pool's edge. Also, microbial community composition appeared to have little correlation with the type of sinter morphology. The number of cell morphotypes identified by fluorescence in situ hybridization and scanning electron microscopy was greater than the number of phylotypes in SSU clone libraries. Despite little variation in Thermocrinis ruber SSU sequences, abundant variation was found in the hypervariable ITS region. The distribution of ITS sequence types appeared to be correlated with distinct morphotypes of Thermocrinis ruber in different pools. Therefore, species- or subspecies-level divergences are present but not detectable in highly conserved SSU sequences.     

Primary sequence determinants responsible for site-selective dephosphorylation of the PDGF beta-receptor by the receptor-like protein tyrosine phosphatase DEP-1

Site-selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor-like protein tyrosine phosphatase DEP-1 site-selectively dephosphorylates the PDGF beta-receptor. DEP-1 dephosphorylation of original and chimeric phospho-peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at -4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position -1 and a preference against a basic residue at position +3. DEP-1 site-selective dephosphorylation of PDGF beta-receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions -1 and +3.     

A single-chain class II MHC-IgG3 fusion protein inhibits autoimmune arthritis by induction of antigen-specific hyporesponsiveness

T cells play a central role in many autoimmune diseases. A method to specifically target the function of autoreactive T cell clones would avoid the global immunosuppression associated with current therapies. To develop a molecule capable of inhibiting autoreactive T cell responses in vivo, single-chain peptide-I-A-IgG3 fusion proteins were constructed and expressed in both mammalian and insect cells. The fusion proteins were designed with an IgG3 Fc moiety to make them divalent, allowing TCR cross-linking, while lacking FcR binding and costimulation. The fusion proteins stimulated T cell hybridomas in vitro in a peptide-specific, MHC-restricted manner but failed to do so in soluble form. In vivo administration of an I-A(q) fusion protein, containing an immunodominant collagen II peptide, significantly delayed the onset and reduced the severity of collagen-induced arthritis in DBA/1 mice by induction of Ag-specific hyporesponsiveness. Such fusion proteins may be useful to study novel therapeutic approaches for T cell-mediated autoimmune diseases.