Localization of angiotensin converting enzyme (kininase II). I. Preparation of antibody-hemeoctapeptide conjugates.

Antibodies to pig lung angiotensin converting enzyme (kininase II) were conjugated to a heme-octapeptide (8-microperoxidase, 8-MP) derived from cytochrome c. 8-MP, which has only one reactive amine, was coupled to antibody in a two-step procedure using a bifunctional active ester, bis-succinyl succinate. In the first-step, 8-MP-succinyl succinate, a stable compound which can be stored. In a second step, the remaining active ester was used for coupling to reactive amines of the antibody. The conjugate consists of 1.6-2.3 8-MP moieties per antibody. Using these procedures, the formation of complex polymers is avoided. Each molecule of conjugate possesses both immunoreactivity and peroxidatic activity. The conjugate has been used to localize angiotensin converting enzyme along the plasma membrane and associated caveolae of pig aortic endothelial cells in culture.     

Transmembrane peptides used to investigate the homo-oligomeric interface and binding hotspot of latent membrane protein 1

Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here, we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full-length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.     

Characteristics of white-tailed deer visits to cattle farms: implications for disease transmission at the wildlife–livestock interface

Bovine tuberculosis (bTB) is endemic in free-ranging white-tailed deer (Odocoileus virginianus) in MI, USA. Currently, the rates of farm visitation by deer and co-use of forage resources by cattle and deer are poorly understood. To evaluate the extent deer and livestock may share forage resources, we investigated farm, yard, and cattle-use area visitation by white-tailed deer and compared visitation with common livestock management practices. We fitted 25 female white-tailed deer near the bTB-infected zone in Michigan’s Lower Peninsula with global positioning system collars. Livestock management practices associated with farm visitation included presence of confined feeding pastures, number of cattle water sources, and the number of cattle pastures. Fewer farm visits occurred at night than during the day. A higher proportion of nighttime visits occurred between midnight and sunrise. Visitation to yards and cattle-use areas were similar: a higher proportion of visits occurred at night, and a higher proportion of nighttime visits occurred between midnight and sunrise. Multiple visits during the same day were common. Visitation increased through spring and peaked during the fawning season. Results suggest that mitigation and control efforts to guard against potential transmission of bTB should include the season and time of day during which deer visitation occurs. Furthermore, specific livestock management practices may contribute to farm visitation by deer. Deer visiting multiple farms may contribute to local area spread of bTB. Focusing risk mitigation efforts on individual deer that are most likely to visit farms may reduce potential bTB transmission.     

A dominant conformational role for amino acid diversity in minimalist protein-protein interfaces

Recent studies have shown that highly simplified interaction surfaces consisting of combinations of just two amino acids, Tyr and Ser, exhibit high affinity and specificity. The high functional levels of such minimalist interfaces might thus indicate small contributions of greater amino acid diversity seen in natural interfaces. Toward addressing this issue, we have produced a pair of binding proteins built on the fibronectin type III scaffold, termed “monobodies.” One monobody contains the Tyr/Ser binary-code interface (termed YS) and the other contains an expanded amino acid diversity interface (YSX), but both bind to an identical target, maltose-binding protein. The YSX monobody bound with higher affinity, a slower off rate and a more favorable enthalpic contribution than the YS monobody. High-resolution X-ray crystal structures revealed that both proteins bound to an essentially identical epitope, providing a unique opportunity to directly investigate the role of amino acid diversity in a protein interaction interface. Surprisingly, Tyr still dominates the YSX paratope and the additional amino acid types are primarily used to conformationally optimize contacts made by tyrosines. Scanning mutagenesis showed that while all contacting Tyr side chains are essential in the YS monobody, the YSX interface was more tolerant to mutations. These results suggest that the conformational, not chemical, diversity of additional types of amino acids provided higher functionality and evolutionary robustness, supporting the dominant role of Tyr and the importance of conformational diversity in forming protein interaction interfaces.     

Observation of a distinct transition in the mode of interconversion of ring pucker conformers in non-crystalline d-ribose-2'-d from 2H NMR spin-alignment

Internal motions of d-ribose selectively ²H-labeled at the 2 position were measured using solid state ²H NMR experiments. A sample of d-ribose-2 -d was prepared in a hydrated, non-crystalline state to eliminate effects of crystal-packing. Between temperatures of –74 and –60°C the C2–H2 bond was observed to undergo two kinds of motions which were similar to those of C2–H2/H2 found previously in crystalline deoxythymidine (Hiyama et al. (1989) J. Am. Chem. Soc., 111, 8609–8613): (1) Nanosecond motion of small angular displacement with an apparent activation energy of 3.6 ± 0.7 kcal mol^–1, and (2) millisecond to microsecond motion of large amplitude with an apparent activation energy 4 kcal mol^–1. At –74°C, the slow, large-amplitude motion was best characterized as a two-site jump with a correlation time on the millisecond time scale, whereas at –60°C it was diffusive on the microsecond time scale. The slow, large-amplitude motions of the C2–H2 bond are most likely from interconversions between C2-endo and C3-endo by way of the O4-endo conformation, whereas the fast, small-amplitude motions are probably librations of the C2–H2 bond within the C2-endo and C3-endo potential energy minima.     

Programmed Cellular Necrosis Mediated by the Pore-Forming α-Toxin from Clostridium septicum

Programmed necrosis is a mechanism of cell death that has been described for neuronal excitotoxicity and ischemia/reperfusion injury, but has not been extensively studied in the context of exposure to bacterial exotoxins. The α-toxin of Clostridium septicum is a β-barrel pore-forming toxin and a potent cytotoxin; however, the mechanism by which it induces cell death has not been elucidated in detail. We report that α-toxin formed Ca²⁺-permeable pores in murine myoblast cells, leading to an increase in intracellular Ca²⁺ levels. This Ca²⁺ influx did not induce apoptosis, as has been described for other small pore-forming toxins, but a cascade of events consistent with programmed necrosis. Ca²⁺ influx was associated with calpain activation and release of cathepsins from lysosomes. We also observed deregulation of mitochondrial activity, leading to increased ROS levels, and dramatically reduced levels of ATP. Finally, the immunostimulatory histone binding protein HMGB1 was found to be released from the nuclei of α-toxin-treated cells. Collectively, these data show that α-toxin initiates a multifaceted necrotic cell death response that is consistent with its essential role in C. septicum-mediated myonecrosis and sepsis. We postulate that cellular intoxication with pore-forming toxins may be a major mechanism by which programmed necrosis is induced.     

Secondary reduction of preference for the sword ornament in the pygmy swordtail Xiphophorus nigrensis (Pisces: Poeciliidae)

Female mating preferences can be secondarily lost for a number of reasons. We examined the preference of female pygmy swordtails, Xiphophorus nigrensis, for the sword, a conspicuous extension of the caudal fin in some males. Females failed to show a preference for conspecific males with swords when presented with live males naturally varying in sword length, with live males of manipulated sword length, and with synthetic animations of males expressing natural variation in sword length. Females showed a significant bias against swords when presented with synthetic animations bearing supernormal sword characteristics. The reduced preference for swords, relative to closely related fish, may result from an increase in the cost of choice due to predation risk, selection against mating with heterospecifics, or changes in the spatial and contrast properties of the conspecific signal.     

Annual climate in Mexican Spotted Owl habitat in the Sacramento Mountains,New Mexico: implications for responding to climate change

Global climate change presents a growing conservation threat, but our understanding of the effects of climate change remains limited for most species. We evaluated the annual climate cycle for threatened Mexican Spotted Owls (Strix occidentalis lucida) in high-elevation mixed-conifer forests in the Sacramento Mountains of New Mexico from 2005 to 2010. We used data from a network of weather stations in Mexican Spotted Owl habitat to describe annual temperature cycles, and precipitation data from a National Weather Service weather station to describe the annual precipitation cycle. We coupled these data with equations from the literature to estimate annual cycles in resting metabolic rate and evaporative water loss, and evaluated the potential effects of a warming climate on these parameters. Annual weather was characterized by cold, dry winters, warmer and dry springs, warm and wet summers, and cool and relatively wet falls. Ambient temperature never exceeded the upper critical temperature for Mexican Spotted Owls (35.2°C), but > 90% of 663,422 hourly temperature observations were below the lower critical temperature (18.2°C). Thus, heat stress was not predicted to occur, but owls likely expended energy on thermoregulation at low temperatures. Resting energy use peaked during winter (December–February) and was lowest when owls would be feeding young (May–August). In contrast, evaporative water loss peaked from June to August, when precipitation also peaked. Mexican Spotted Owls generally appeared well-adapted to the current climate cycle in our montane study area, but late winter (February–March) may be a critical period in terms of energy requirements, and late spring (April–May) may be critical in terms of water relationships. Predicted changes in temperature through 2099 would result in reductions in predicted energy use by Mexican Spotted Owls, but increases in predicted water use. Water relationships may become increasingly important for Mexican Spotted Owls in the face of climate change, especially in warmer and drier areas. In forested areas, retaining patches of older forest with high canopy cover in cool, mesic sites may provide continued benefits to Mexican Spotted Owls under climate change.