Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Interaction between corticosteroid binding globulin and activated leukocytes in vitro

The interaction between human corticosteroid binding globulin and activated leukocytes is restricted to the granulocyte population, and is characterized by specific proteolytic cleavage of corticosteroid binding globulin which markedly reduces its steroid binding activity. A direct interaction between corticosteroid binding globulin and the activated cells appears to enhance this event, and does not involve cellular internalization of corticosteroid binding globulin or its proteolytic degradation products, which resemble those obtained after incubation of corticosteroid binding globulin with neutrophil elastase. These data suggest that corticosteroid binding globulin interacts with elastase on the surface of activated neutrophils, and may promote glucocorticoid delivery to these cells during inflammation.     

AN OCEAN-BASIN-WIDE MARK-RECAPTURE STUDY OF THE NORTH ATLANTIC HUMPBACK WHALE (MEGAPTERA NOVAEANGLIAE)

Although much is known about the humpback whale, Megaptera novaeangliae, regional studies have been unable to answer several questions that are central to the conservation and management of this endangered species. To resolve uncertainties about population size, as well as the spatial and genetic structure of the humpback whale population in the North Atlantic, we conducted a two-year ocean-basin-wide photographic and biopsy study in 1992-1993. Photographic and skin-biopsy sampling was conducted of animals in feeding and breeding areas throughout most of the range of this species in the North Atlantic, from the West Indies breeding grounds through all known feeding areas as far north as arctic Norway. A standardized sampling protocol was designed to maximize sample sizes while attempting to ensure equal probability of sampling, so that estimates of abundance would be as accurate and as precise as possible. During 666 d at sea aboard 28 vessels, 4,207 tail fluke photographs and 2,326 skin biopsies were collected. Molecular analyses of all biopsies included determination of sex, genotype using six microsatellite loci, and mitochondrial control region sequence. The photographs and microsatellite loci were used to identify 2,998 and 2,015 individual whales, respectively. Previously published results from this study have addressed spatial distribution, migration, and genetic relationships. Here, we present new estimates of total abundance in this ocean using photographic data, as well as overall and sex-specific estimates using biopsy data. We identify several potential sampling biases using only breeding-area samples and report a consistent mark-recapture estimate of oceanwide abundance derived from photographic identification, using both breeding and feeding-area data, of 10,600 (95% confidence interval 9,300-12,100). We also report a comparable, but less precise, biopsy-based estimate of 10,400 (95% confidence interval of 8,000-13,600). These estimates are significantly larger and more precise than estimates made for the 1980s, potentially reflecting population growth. In contrast, significantly lower and less consistent estimates were obtained using between-feeding-area or between-breeding-area sampling. Reasons for the lower estimates using the results of sampling in the same areas in subsequent years are discussed. Overall, the results of this ocean-basin-wide study demonstrate that an oceanwide approach to population assessment of baleen whales is practicable and results in a more comprehensive understanding of population abundance and biology than can be gained from smaller-scale efforts.     

Development of monoclonal antibodies that identify Vibrio species commonly isolated from infections of humans, fish, and shellfish.

Monoclonal antibodies (MAbs) against Vibrio species that infect humans, fish, and shellfish were developed for application in rapid identifications. The pathogens included Vibrio alginolyticus, V. anguillarum, V. carchariae, V. cholerae, V. damsela, V. furnissii, V. harveyi, V. ordalii, V. parahaemolyticus, and V. vulnificus. Three types of MAbs were selected. The first important group included MAbs that reacted with only a single species. A second group comprised a number of MAbs that reacted with two, taxonomically closely related Vibrio species. For example, of 22 MAbs raised against V. alginolyticus, 6 recognized a 52-kDa flagellar H antigen common to both V. alginolyticus and V. parahaemolyticus; V. anguillarum and V. ordalii also shared antigens. A third group included three genus-specific MAbs that reacted with almost all Vibrio species but did not react with other members of the family Vibrionaceae (e.g., members of the Aeromonas, Photobacterium, and Plesiomonas genera) or a wide range of gram-negative bacteria representing many genera. This last group indicated the possible existence of an antigenic determinant common to Vibrio species. Two of these three genus-specific MAbs reacted with heat-stable antigenic determinants of Vibrio species as well as lipopolysaccharide extracted from Vibrio species. The use of the MAbs in blind tests and diagnosis of clinical isolates indicated that three different types of bacteria, viz., live, formalin-fixed, and sodium azide-killed bacteria, were detected consistently. Overall, it was found that the genus-specific MAbs were very useful for rapidly identifying vibrios in the screening of acute infections, while the species-specific MAbs and others were useful for completing the diagnosis.