Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.     

Primary Kaposi's sarcoma of an intraparotid lymph node with AIDS

A case of Kaposi's sarcoma of an intraparotid lymph node in a patient with previously undiagnosed AIDS is presented. In patients at risk for AIDS who present with undiagnosed head and neck tumors, the diagnosis of epidemic Kaposi's sarcoma should be considered. Although transmission of AIDS to health care workers is exceedingly rare, proper precautions should be exercised when working with these patients.     

Molecular evolution of the maize sex-determining gene TASSELSEED2 in Bouteloua (Poaceae)

The majority of angiosperms produce hermaphrodite flowers, while a lesser number (20–30%) produce unisexual flowers. Little is known about the molecular biology of sex-determination in angiosperms, however, a few sex-determining genes have been cloned from the model system Zea mays. One of these genes is Tasselseed2 (Ts2) which has been shown to be involved in the arrest of developing pistils in male flowers. In this study, we sequenced a putative homologue of Ts2 in species of Bouteloua, a genus in the grass subfamily Chloridoideae. We found significant genetic variation at Ts2 in Bouteloua relative to other developmental genes characterized in maize and other grass species. We also found that in Bouteluoua, Ts2 is evolving non-neutrally in the hermaphrodite-flowered Bouteloua hirsuta while no difference from neutral expectation was detected at Ts2 in the monoecious/dioecious Bouteloua dimorpha. The putatively neutral gene Alcohol Dehydrogenase1 (Adh1) was also examined for the same species of Bouteloua, and no departure from neutral expectation was detected. Our results suggest that purifying selection may be acting on Ts2 in the hermaphrodite-flowered B. hirsuta while no evidence of selection was detected at Ts2 in the monoecious/dioecious B. dimorpha.     

Functional amino acid supplementation postweaning mitigates the response of normal birth weight more than for low birth weight pigs to a subsequent Salmonella challenge

Previous work has shown that dietary supplementation with key functional amino acids (FAA) improves growth performance and immune status of disease-challenged normal birth weight (NBW) pigs. It is not known whether FAA supplementation attenuates the effects of a subsequent disease challenge or whether this response is similar in low birth weight (LBW) pigs. The objective was to determine the effects of birth weight and FAA supplementation during the postweaning period in Salmonella-challenged pigs. Thirty-two LBW (1.08 ± 0.11 kg) and NBW (1.58 ± 0.11 kg) pigs were assigned to a nursery feeding program at weaning (25 d) for 31 days in a 2 × 2 factorial arrangement. Factors were birth weight category (LBW vs. NBW) and basal (FAA–) or supplemented FAA profile (FAA+; Thr, Met, and Trp at 120% of requirements). At d 31, pigs were placed onto a common grower diet and, after a 7-d adaptation period, were inoculated with Salmonella Typhimurium (ST; 2.2 × 10⁹ colony-forming units/mL) and monitored for 7-d postinoculation. Growth performance, rectal temperature, fecal score, indicators of gut health, ST shedding score in feces, intestinal ST colonization and translocation, and blood parameters of acute-phase response and antioxidant balance were measured pre- and postinoculation. Inoculation with ST increased temperature and fecal score, and the overall rectal temperature was higher in LBW compared to NBW pigs (P < 0.05). Postinoculation (d 7), reduced:oxidized glutathione was increased in NBW compared to LBW pigs (P < 0.05). Salmonella shedding and translocation to spleen were lower in NBW-FAA+ compared to NBW-FAA? pigs (P < 0.05). Postinoculation average daily gain was higher in NBW-FAA+ (P < 0.05) compared to the other groups. Postinoculation haptoglobin, superoxide dismutase, and colonic myeloperoxidase were increased in LBW-FAA? pigs (P < 0.05). Ileal alkaline phosphatase was decreased in LBW compared to NBW (P < 0.05). Overall, FAA supplementation represents a potential strategy to mitigate the effect of enteric disease challenge in NBW, but not LBW pigs.     

Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.     

The effect of ethylene glycol on the phytovolatilization of 1,4-dioxane

Phytoremediation at contaminated sites is often complicated by the presence of more than one chemical However, the effects of common co-contaminants such as ethylene glycol on the phytoremediation of other chemicals, e.g., 1,4-dioxane, is not well understood. Field studies with DN34 poplar trees revealed a 28% decline in growth rate in response to 10 g/L ethylene glycol in the groundwater, thus indicating a significant and deleterious effect on tree viability, and likely, the plants' utility for phytoremediation. Thorough investigations using Arabidopsis thaliana, with its small size and rapid life cycle, indicated significant growth reduction at 10 g/L and complete inhibition of germination at 40 g/L ethylene glycol Ethylene glycol was almost as severe a stressor as the well characterized osmotic inhibitor, sorbitoL Watering potted trees with 10 g/L ethylene glycol reduced their growth by more than 50%, and similar results were observed in hydroponically grown poplar and willow trees. Under hydroponic conditions, 60 g/L ethylene glycol inhibited the phytovolatilization of l,4-dioxane by more than 80%, and all trees evapo-transpired 1,4-dioxane less efficiently than water. In fact, this efficiency differed between trees and the difference became more pronounced in the presence of ethylene glycol.     

Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.     

Expression, purification, and characterization of Thermotoga maritima membrane proteins for structure determination

Structural studies of integral membrane proteins typically rely upon detergent micelles as faithful mimics of the native lipid bilayer. Therefore, membrane protein structure determination would be greatly facilitated by biophysical techniques that are capable of evaluating and assessing the fold and oligomeric state of these proteins solubilized in detergent micelles. In this study, an approach to the characterization of detergent-solubilized integral membrane proteins is presented. Eight Thermotoga maritima membrane proteins were screened for solubility in 11 detergents, and the resulting soluble protein-detergent complexes were characterized with small angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD) spectroscopy, and chemical cross-linking to evaluate the homogeneity, oligomeric state, radius of gyration, and overall fold. A new application of SAXS is presented, which does not require density matching, and NMR methods, typically used to evaluate soluble proteins, are successfully applied to detergent-solubilized membrane proteins. Although detergents with longer alkyl chains solubilized the most proteins, further characterization indicates that some of these protein-detergent complexes are not well suited for NMR structure determination due to conformational exchange and protein oligomerization. These results emphasize the need to screen several different detergents and to characterize the protein-detergent complex in order to pursue structural studies. Finally, the physical characterization of the protein-detergent complexes indicates optimal solution conditions for further structural studies for three of the eight overexpressed membrane proteins.     

C3 photosynthesis in Aristida longifolia: Implication for photosynthetic diversification in Aristidoideae (Poaceae)

Only a small percentage of plant species undergo C(4) photosynthesis. Despite its rarity, the C(4) pathway has evolved numerous times from C(3) ancestors, with as many as 18 independent origins in grasses alone. We report non-Kranz (C(3)) anatomy in Aristida longifolia, a species in a genus of ca. 300 species previously thought to possess only Kranz (C(4)) anatomy. Leaf blade transections of A. longifolia show widely spaced vascular bundles, nonradiate chlorenchyma, and few or no chloroplasts in cells of the sheaths surrounding the vascular bundle, all features indicative of C(3) photosynthesis. Carbon isotope ratios range from -27.68 to -29.71%, likewise indicative of C(3) photosynthesis. We also reconstruct the phylogeny of Aristidoideae, comprising Aristida, Sartidia (C(3)), and Stipagrostis (C(4)), using a sample of 11 species, including A. longifolia, and DNA sequences of the nuclear ribosomal internal transcribed spacer region and the chloroplast rpl16 intron and trnL-trnF region. Sartidia and Stipagrostis resolve as sisters, and sister to this clade is Aristida. Aristida longifolia resolves as sister to the remaining species in the genus. C(3) photosynthesis is hypothesized to be ancestral in Aristidoideae, which means the C(4) pathway evolved twice in the subfamily-in Stipagrostis and early in the diversification of the Aristida clade.