Helical packing patterns in membrane and soluble proteins

This article presents the results of a detailed analysis of helix-helix interactions in membrane and soluble proteins. A data set of interacting pairs of helices in membrane proteins of known structure was constructed and a structure alignment algorithm was used to identify pairs of helices in soluble proteins that superimpose well with pairs of helices in the membrane-protein data set. Most helix pairs in membrane proteins are found to have a significant number of structural homologs in soluble proteins, although in some cases, primarily involving irregular helices, no close homologs exist. An analysis of geometric relationships between interacting helices in the two sets of proteins identifies some differences in the distributions of helix length, interfacial area, packing angle, and distance between the polypeptide backbones. However, a subset of soluble-protein helix pairs that are close structural homologs to membrane-protein helix pairs exhibits distributions that mirror those observed in membrane proteins. The larger average interface size and smaller distance of closest approach seen for helices in membrane proteins appears due in part to a relative enrichment of alanines and glycines, particularly as components of the AxxxA and GxxxG motifs. It is argued that membrane helices are not on average more tightly packed than helices in soluble proteins; they are simply able to approach each other more closely. This enables them to interact over longer distances, which may in turn facilitate their remaining in contact over much of the width of the lipid bilayer. The close structural similarity seen between some pairs of helices in membrane and soluble proteins suggests that packing patterns observed in soluble proteins may be useful in the modeling of membrane proteins. Moreover, there do not appear to be fundamental differences between the magnitude of the forces that drive helix packing in membrane and soluble proteins, suggesting that strategies to make membrane proteins more soluble by mutating surface residues are likely to encounter success, at least in some cases.     

Markov chain analysis of succession in a rocky subtidal community

We present a Markov chain model of succession in a rocky subtidal community based on a long-term (1986-1994) study of subtidal invertebrates (14 species) at Ammen Rock Pinnacle in the Gulf of Maine. The model describes successional processes (disturbance, colonization, species persistence, and replacement), the equilibrium (stationary) community, and the rate of convergence. We described successional dynamics by species turnover rates, recurrence times, and the entropy of the transition matrix. We used perturbation analysis to quantify the response of diversity to successional rates and species removals. The equilibrium community was dominated by an encrusting sponge (Hymedesmia) and a bryozoan (Crisia eburnea). The equilibrium structure explained 98% of the variance in observed species frequencies. Dominant species have low probabilities of disturbance and high rates of colonization and persistence. On average, species turn over every 3.4 years. Recurrence times varied among species (7-268 years); rare species had the longest recurrence times. The community converged to equilibrium quickly (9.5 years), as measured by Dobrushin's coefficient of ergodicity. The largest changes in evenness would result from removal of the dominant sponge Hymedesmia. Subdominant species appear to increase evenness by slowing the dominance of Hymedesmia. Comparison of the subtidal community with intertidal and coral reef communities revealed that disturbance rates are an order of magnitude higher in coral reef than in rocky intertidal and subtidal communities. Colonization rates and turnover times, however, are lowest and longest in coral reefs, highest and shortest in intertidal communities, and intermediate in subtidal communities.     

Phylogenetic position and biogeography of Hillebrandia sandwicensis (Begoniaceae): a rare Hawaiian relict

The Begoniaceae consist of two genera, Begonia, with approximately 1400 species that are widely distributed in the tropics, and Hillebrandia, with one species that is endemic to the Hawaiian Islands and the only member of the family native to those islands. To help explain the history of Hillebrandia on the Hawaiian Archipelago, phylogenetic relationships of the Begoniaceae and the Cucurbitales were inferred using sequence data from 18S, rbcL, and ITS, and the minimal age of both Begonia and the Begoniaceae were indirectly estimated. The analyses strongly support the placement of Hillebrandia as the sister group to the rest of the Begoniaceae and indicate that the Hillebrandia lineage is at least 51-65 million years old, an age that predates the current Hawaiian Islands by about 20 million years. Evidence that Hillebrandia sandwicensis has survived on the Hawaiian Archipelago by island hopping from older, now denuded islands to younger, more mountainous islands is presented. Various scenarios for the origin of ancestor to Hillebrandia are considered. The geographic origin of source populations unfortunately remains obscure; however, we suggest a boreotropic or a Malesian-Pacific origin is most likely. Hillebrandia represents the first example in the well-studied Hawaiian flora of a relict genus.     

A stochastic model of cytotoxic T cell responses

We have constructed a stochastic stage-structured model of the cytotoxic T lymphocyte (CTL) response to antigen and the maintenance of immunological memory. The model follows the dynamics of a viral infection and the stimulation, proliferation, and differentiation of na?ve CD8(+) T cells into effector CTL, which can eliminate virally infected cells. The model is capable of following the dynamics of multiple T cell clones, each with a T cell receptor represented by a digit string. MHC-viral peptide complexes are also represented by strings and a string match rule is used to compute the affinity of a T cell receptor for a viral epitope. The avidities of interactions are also computed by taking into consideration the density of MHC-viral peptides on the surface of an infected cell. Lastly, the model allows the probability of T cell stimulation to depend on avidity but also incorporates the notion of an antigen-independent programmed proliferative response. We compare the model to experimental data on the cytotoxic T cell response to lymphocytic choriomeningitis virus infections.     

Virus Shedding and Potential for Interspecies Waterborne Transmission of Highly Pathogenic H5N1 Influenza Virus in Sparrows and Chickens

To elucidate the role of sparrows as intermediate hosts of highly pathogenic avian influenza H5N1 viruses, we assessed shedding and interspecies waterborne transmission of A/duck/Laos/25/06 in sparrows and chickens. Inoculated birds shed virus at high titers from the oropharynx and cloaca, and infection was fatal. Waterborne transmission from inoculated sparrows to contact chickens was absent, while 25% of sparrows were infected via waterborne transmission from chickens. The viral shedding and susceptibility to infection we observed in sparrows, coupled with their presence in poultry houses, could facilitate virus spread among poultry and wild birds in the face of an H5N1 influenza virus outbreak.The H5N1 influenza A viruses remain a major global concern because of their rapid evolution, genetic diversity, broad host range, and ongoing circulation in wild and domestic birds. H5N1 influenza viruses have swept through poultry flocks across Asia and have spread westward through Eastern Europe to India and Africa since 2003 (1). Sixty-two countries have reported H5N1 influenza virus in domestic poultry/wild birds during the time period 2003 to 2009 (http://www.oie.int/eng/info_ev/en_AI_factoids_2.htm), and to date, more than 400 human infections have been documented in 16 countries, with a mortality rate of ∼61% (http://www.who.int/csr/disease/avian_influenza/country/cases_table_2009_05_22/en/index.html). Most human cases of H5N1 influenza have occurred after contact with infected poultry (13).Some of the more recent isolates of H5N1 highly pathogenic avian influenza (HPAI) virus do not cause overt disease in certain species of domestic and wild ducks; however, these viruses are 100% lethal to chickens and gallinaceous poultry. Because of ducks’ ability to “silently” spread H5N1 HPAI virus and their unresolved role as a reservoir, they are the focus of much research (5, 6, 11). In contrast, the possible role of passerine birds has received little attention, despite their widespread interaction with poultry at many sites worldwide (http://www.searo.who.int/LinkFiles/Publication_PHI-prevention-control-AI.pdf). The order Passeriformes includes more than half of all bird species, including sparrows. Since 2001, several outbreaks of H5N1 influenza virus infection have been reported in passerine birds in eastern Asia, often near infected poultry farms (15). Interestingly, the only confirmed presence of asymptomatic infection with HPAI H5N1 in wild birds was in tree sparrows in Henan Province, China. Both tree and house sparrows (Passer montanus and Passer domesticus, respectively) are members of the Old World sparrow family Passeridae, and in fact, the tree sparrow was not recognized as a species separate from that of the house sparrow until 1713 (http://www.arkive.org/tree-sparrow/passer-montanus/info.html?displayMode=factsheet). The four avian influenza virus isolates obtained from these asymptomatic infections were of the A/Goose/Guangdong/1/96 lineage and were highly pathogenic to experimentally infected chickens (4, 8).Under experimental conditions, passerine species have shown varied susceptibility to HPAI H5N1 viruses. Among sparrows, starlings, and pigeons inoculated with HPAI H5N1 virus isolates, only sparrows experienced lethal infection, and transmission to contact birds was extremely rare (2). Similarly, in sparrows and starlings inoculated with the H5N1 HPAI A/chicken/Hong Kong/220/97 virus, clinical signs were observed only in sparrows, and no deaths occurred (9).To assess the duration and routes of virus shedding and the waterborne virus transmission of HPAI H5N1 virus between sparrows and chickens, we inoculated groups of birds with A/duck/Laos/25/06, which had caused extremely high morbidity and mortality in domestic ducks (7) and was highly pathogenic to chickens, geese, and quail (J.-K. Kim and R. G. Webster, unpublished data). The virus was obtained from our collaborators in Lao People''s Democratic Republic and was grown in the allantoic cavities of 10-day-old embryonated chicken eggs (eggs) for 36 to 48 h at 35°C. The allantoic fluid was harvested, titrated (50% egg infective dose [EID₅₀] per milliliter), and stored at −80°C. All experiments were approved by the U.S. Department of Agriculture and the U.S. Centers for Disease Control and Prevention and were performed in biosafety level 3+ facilities at St. Jude Children''s Research Hospital. Wild house sparrows (Passer domesticus) were captured locally (Memphis, TN), and specific-pathogen-free outbred White Leghorn chickens (Gallus domesticus) were purchased from Charles River Laboratories (North Franklin, CT). All animal experiments were approved by the St. Jude Animal Care and Use Committee and complied with the policies of the National Institutes of Health and the Animal Welfare Act.Before inoculation, oropharyngeal and cloacal swabs were collected from sparrows, and baseline blood samples were collected from chickens to exclude preexisting H5N1 influenza virus infection. Eight sparrows were inoculated intranasally with 10⁶ EID₅₀ of virus in a volume of 100 μl, and five chickens were inoculated with 10² EID₅₀ of virus in a volume of 1 ml (0.5 ml intranasally, 0.5 ml intratracheally, and 1 drop per eye). All birds in each experimental group were housed in a single cage. Inoculated sparrows were provided with 1 liter of water in a shallow stainless steel pan at the bottom of the cage, and chickens were given 3 liters of water in a trough inside the cage. Twenty-four hours after inoculation, 1 liter of water was removed from the inoculated chickens’ cage and placed undiluted in a cage housing 8 contact sparrows; similarly, 1 liter of water was taken from the inoculated sparrows’ cage, mixed with 2 liters of fresh water, and placed in a cage housing 5 contact chickens. Clinical disease signs, including depression, huddling at the cage bottom, and ruffled feathers, were monitored through daily observation, and oropharyngeal and cloacal swabs obtained from all birds were collected daily for 14 days. Swab samples were titrated in eggs and expressed as log₁₀ EID₅₀/ml (10). The lower limit of detection was 0.75 log₁₀ EID₅₀/ml.Blood samples were taken from all surviving contact birds on day 14 of the study. Sera were treated with a receptor-destroying enzyme (Denka Seiken, Campbell, CA), as instructed by the manufacturer, and heat inactivated at 56°C for 30 min. Hemagglutination inhibition (HI; using 0.5% packed chicken red blood cells) titers were determined as the reciprocal of the highest serum dilution that inhibited 4 hemagglutinating units of virus. HI titers of ≥10 were considered suggestive of recent influenza virus infection.Inoculation with A/duck/Laos/25/06 was lethal to all birds (Table ​(Table1).1). While chickens succumbed to infection within 2 days postinoculation (p.i.), the mean time until death for sparrows was 4.1 days; mortality occurred rapidly (overnight) without prior observation of clinical signs. Expected clinical signs, should they have occurred, included moderate to severe depression, huddling at the cage bottom, and ruffled feathers (9). All inoculated birds shed virus from the oropharynx and, to a lesser extent, from the cloaca (Fig. 1A and B). The mean virus titers of inoculated chickens and sparrows were comparable on day 1 p.i.; however, on day 2 p.i., the mean oropharyngeal and cloacal viral titers of chickens were approximately 2 and 2.5 times greater, respectively, than those of sparrows (Fig. 1A and B). The virus titer in water used by inoculated sparrows was 10^0.75 EID₅₀/ml at 1 day p.i. and peaked at 10^1.75 EID₅₀/ml on days 2 and 4 p.i. (Fig. ​(Fig.1C).1C). No virus was detected in water from the inoculated chickens’ cage.Open in a separate windowFIG. 1.Mean oropharyngeal and cloacal virus titers in sparrows (A) and chickens (B) inoculated with a lethal dose of A/duck/Laos/25/06 (H5N1) virus. (C) Virus titers in the drinking water of inoculated sparrows. Sparrows were inoculated with 10⁶ EID₅₀/ml of virus, and chickens were inoculated with 10² EID₅₀/ml of virus. The lower limit of detection was 0.75 log₁₀ EID₅₀/ml.

TABLE 1.

Transmission rates, mortality rates, and mean peak titers of A/duck/Laos/25/06 (H5N1) virus in inoculated and contact birds

Simultaneous monitoring of electroformation of phospholipid vesicles by quartz crystal microbalance and optical microscopy

The electroformation of giant vesicles from 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) was monitored using quartz crystal microbalance with dissipation monitoring (QCM-D) and optical microscopy, simultaneously using a novel sample cell design. A gold-coated QCM crystal was used as one of the electrodes and an Indium–tin-oxide (ITO)-coated glass slide was used as the second electrode for electroformation. Increases in the frequency and decreases in the dissipation were observed immediately upon voltage application between the two electrodes, indicating the loss of lipid from the QCM surface. Concurrently, we observed vesicles on the QCM electrode surface by differential interference contrast (DIC)-optical microscopy. The lipid-coated substrates were measured with AFM at various stages in the electroformation, and a significant change in the morphology of the lipid film was observed. Ellipsometry was used to find the average thickness of lipid film. The QCM data were fitted to a viscoelastic model to determine the viscoelastic properties and time dependence of the film thickness. All methods used to determine film thickness give values in reasonable quantitative agreement. Differences between the methods are consistent with what one might expect due to what is actually measured in the individual techniques. The comparison between mass loss and observed vesicles suggest that the vesicles formed are first localized to the substrate and then slowly released into the solution. By comparing the mass lost from the lipid film, to the total surface area of lipid vesicles observed, it is apparent that only a relatively small fraction of the lipid goes into the production of unilamellar vesicles with sizes detectable with optical microscopy.     

Neural regulation of slow-wave frequency in the murine gastric antrum

Gastric peristaltic contractions are driven by electrical slow waves modulated by neural and humoral inputs. Excitatory neural input comes primarily from cholinergic motor neurons, but ACh causes depolarization and chronotropic effects that might disrupt the normal proximal-to-distal spread of gastric slow waves. We used intracellular electrical recording techniques to study cholinergic responses in stomach tissues from wild-type and W/W(V) mice. Electrical field stimulation (5 Hz) enhanced slow-wave frequency. These effects were abolished by atropine and the muscarinic M(3)-receptor antagonist 4-diphenylacetoxy-N-methylpiperidine methiodide. ACh released from nerves did not depolarize antral muscles. At higher rates of stimulation (10 Hz), chronotropic effects were mediated by ACh and a noncholinergic transmitter and blocked by muscarinic antagonists and neurokinin (NK(1) and NK(2))-receptor antagonists. Neostigmine enhanced slow-wave frequency, suggesting that the frequency of antral pacemakers is kept low by efficient metabolism of ACh. Neostigmine had no effect on slow-wave frequency in muscles of W/W(v) mice, which lack intramuscular interstitial cells of Cajal (ICC-IM). These muscles also showed no significant chronotropic response to 5-Hz electrical field stimulation or the cholinergic agonist carbachol. The data suggest that the chronotropic effects of cholinergic nerve stimulation occur via ICC-IM in the murine stomach. The capacity of gastric muscles to metabolize ACh released from enteric motor neurons contributes to the maintenance of the proximal-to-distal slow-wave frequency gradient in the murine stomach. ICC-IM play a critical role in neural regulation of gastric motility, and ICC-IM become the dominant pacemaker cells during sustained cholinergic drive.     

Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle

The objective of the present study was to determine differences in time of detection of pregnancy between heifers and cows and the interval after insemination at which the maximum sensitivity and negative predictive value of transrectal ultrasonography were obtained. One-thousand-four-hundred transrectal ultrasonographies (TRUS-1; 1,079 in cows and 321 in heifers) were performed using a 5-MHz linear-array transducer. The cattle were randomly assigned to have TRUS performed once between days 24 and 30 (estrus=day 0) in cows or between days 21 and 27 in heifers. Every TRUS diagnosis was subsequently compared with a second TRUS diagnosis (TRUS-2), performed 3-8 days later, after day 30 (range 31-38) for cows and after day 27 (range 28-35) for heifers. The sensitivity and specificity between cows and heifers for the common days of TRUS (from 24 to 27) were compared. In cows, sensitivity increased gradually from 74.5% at day 24 to 100% at day 29 (P<0.01). Specificity increased from days 24-25 and reached a plateau of 96.6% on day 26 (P<0.01). In heifers, sensitivity increased from 50% at day 21 to 100% at day 26 (P<0.01). Specificity increased from 87.5% at day 21 and remained steady at 94% starting on day 23 (P>0.05). The sensitivity for cows and heifers was 89.2 and 96.8%, respectively (P<0.05) and the specificity was 93.0 and 93.4% (P>0.05). In this study, heifers were diagnosed pregnant earlier than cows, and the maximum sensitivity and negative predictive value were obtained 3 days earlier in heifers than cows (days 26 and 29, respectively).     

A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex

Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.