Lynne S. Arneson

Correlation of metabolism with tissue carbon and nitrogen turnover rate in small mammals

Stable isotopes have proven to be a useful tool for deciphering food webs, examining migration patterns and determining nutrient resource allocation. In order to increase the descriptive power of isotopes, an increasing number of studies are using them to model tissue turnover. However, these studies have, mostly by necessity, been largely limited to laboratory experiments and the demand for an easier method of estimating tissue turnover in the field for a large variety of organisms remains. In this study, we have determined the turnover rate of blood in mice and rats using stable isotope analysis, and compared these rates to the metabolic rates of the animals. Rats (Rattus norvegicus) (n=4) and mice (Mus musculus) (n=4) were switched between isotopically distinct diets, and the rate of change of δ13C and δ15N in whole blood was determined. Basal metabolic rates (as CO2 output and O2 consumption per unit time, normalized for mass) were determined for the rats and mice. Rats, which were an order of magnitude larger and had a slower metabolic rate per unit mass than mice (0.02 vs. 0.14 O2/min/g), had a slower blood turnover than mice for 13C (t1/2xa0=24.8 and 17.3xa0days, respectively) and 15N (t1/2xa0=27.7 and 15.4xa0days, respectively). A positive correlation between metabolic rate and blood isotopic turnover rate was found. These are the only such data for mammals available, but the literature for birds shows that mass and whole-body metabolic rates in birds scale logarithmically with tissue turnover. Interestingly, the mammalian data graph separately from the bird data on a turnover versus metabolic rate plot. Both mice and rat tissue in this study exhibited a slower turnover rate compared to metabolic rate than for birds. These data suggest that metabolic rate may be used to estimate tissue turnover rate when working with organisms in the field, but that a different relationship between tissue turnover and metabolism may exist for different classes of organisms.

Potent effects of low levels of MHC class II-associated invariant chain on CD4+ T cell development.

Invariant chain (Ii)-negative mice exhibit defects in MHC class II assembly and transport that results in reduced levels of surface class II, altered antigen presentation, and inefficient positive selection of CD4+ T cells. Many CD4+ T cells that do mature in Ii-negative mice express a cell surface phenotype consistent with aberrant positive selection or peripheral activation. Reconstitution of these mice with low levels of either the p31 or p41 form of Ii does not restore transport of the bulk of class II or class II surface expression, but surprisingly does restore positive selection as measured by numbers and surface phenotype of CD4+ T cells. Thus, an Ii-dependent process, independent of effects on class II surface density, appears to be required for normal positive selection of CD4+ T cells.

Hydrogen Bond Integrity Between MHC Class II Molecules and Bound Peptide Determines the Intracellular Fate of MHC Class II Molecules

MHC class II molecules associate with peptides through pocket interactions and the formation of hydrogen bonds. The current paradigm suggests that the interaction of side chains of the peptide with pockets in the class II molecule is responsible for the formation of stable class II-peptide complexes. However, recent evidence has shown that the formation of hydrogen bonds between genetically conserved residues of the class II molecule and the main chain of the peptide contributes profoundly to peptide stability. In this study, we have used I-Ak, a class II molecule known to form strong pocket interactions with bound peptides, to probe the general importance of hydrogen bond integrity in peptide acquisition. Our studies have revealed that abolishing hydrogen bonds contributed by positions 81 or 82 in the β-chain of I-Ak results in class II molecules that are internally degraded when trafficked through proteolytic endosomal compartments. The presence of high-affinity peptides derived from either endogenous or exogenous sources protects the hydrogen bond-deficient variant from intracellular degradation. Together, these data indicate that disruption of the potential to form a complete hydrogen bond network between MHC class II molecules and bound peptides greatly diminishes the ability of class II molecules to bind peptides. The subsequent failure to stably acquire peptides leads to protease sensitivity of empty class II molecules, and thus to proteolytic degradation before export to the surface of APCs.

The chondroitin sulfate form of invariant chain trimerizes with conventional invariant chain and these complexes are rapidly transported from the trans-Golgi network to the cell surface

Targeting of MHCII-invariant chain complexes from the trans-Golgi network to endosomes is mediated by two di-leucine-based signals present in the cytosolic domain of invariant chain. Generation of this endosomal targeting signal is also dependent on multimerization of the invariant chain cytosolic domain sequences, mediated through assembly of invariant chain into homotrimers. A small subset of invariant chain is modified by the addition of chondroitin sulfate and is expressed on the cell surface in association with MHCII. In the present study, we have followed the biosynthetic pathway and route of intracellular transport of this proteoglycan form of invariant chain. We found that the efficiency of chondroitin sulfate modification can be increased by altering the invariant chain amino acid sequence around Ser-201 to the xylosylation consensus sequence. Our results also indicate that, following sulfation, the proteoglycan form is transported rapidly from the trans-Golgi network to the cell surface and is degraded following internalization into an endocytic compartment. Invariant chain-chondroitin sulfate is present in invariant chain trimers that also include conventional non-proteoglycan forms of invariant chain. These data indicate that invariant chain-chondroitin sulfate-containing complexes are transported rapidly from the trans-Golgi network to the cell surface in spite of the presence of an intact endosomal localization signal. Furthermore, these results suggest that invariant chain-chondroitin sulfate may play an important role in the generation of cell-surface pools of invariant chain that can serve as receptors for CD44 and macrophage migration inhibitory factor.

The MHC class II molecule I-Ag7 exists in alternate conformations that are peptide dependent

Insulin-dependent diabetes mellitus is an autoimmune disease that is genetically linked to the HLA class II molecule DQ in humans and to MHC I-Ag7 in nonobese diabetic mice. The I-Ag7 β-chain is unique and contains multiple polymorphisms, at least one of which is shared with DQ alleles linked to insulin-dependent diabetes mellitus. This polymorphism occurs at position 57 in the β-chain, in which aspartic acid is mutated to a serine, a change that results in the loss of an interchain salt bridge between αArg76 and βAsp57 at the periphery of the peptide binding groove. Using mAbs we have identified alternative conformations of I-Ag7 class II molecules. By using an invariant chain construct with various peptides engineered into the class II-associated invariant chain peptide (CLIP) region we have found that formation of these conformations is dependent on the peptide occupying the binding groove. Blocking studies with these Abs indicate that these conformations are present at the cell surface and are capable of interactions with TCRs that result in T cell activation.

The intraspecies relationship between tissue turnover and metabolic rate in rats

Ecologists interested in studying fluctuating relationships between consumers and nutrient sources are increasingly involved in modeling the rate at which consumers incorporate dietary components. In mammals a correlation between resting metabolic rate (RMR) and tissue turnover may exist across a range of species. Less is known about the variation of tissue turnover rate within a species, and how that correlates with RMR. Here we examine two strains of rats (Rattus norvegicus) with different RMR to test whether variation in RMR is positively correlated with tissue turnover rate within a species. If RMR, a relatively simple measurement, can be correlated with tissue turnover, then this relationship could be used to better interpret ecological functions, including impact of migratory or seasonally available nutrient sources. Here, the changing isotope signature in rat whole blood was modeled using a modified exponential decay equation and a reaction progress variable model. The modeled rate of turnover, metabolic rate (O2 consumed), and mass were then compared between strains of rats. The mass and RMRs (conditions during which RMRs were determined modified from the ideal, as outlined in the Methods) were significantly different between strains, but half-life and the metabolic tissue replacement component of turnover (as opposed to turnover from mass gain) were not. No significant correlation was found between RMR and metabolic tissue replacement between the strains. Results suggest that within a species showing a range of RMRs, blood tissue turnover should not vary significantly.