Evidence of leptospirosis in the kidneys and serum of feral swine (Sus scrofa) in the United States.

Leptospirosis is the most widespread zoonosis in humans worldwide. In the United States, widespread detection of antibodies to leptospirosis have been identified in feral swine (Sus scrofa) with the highest detection of serovars, Bratislava, Icterohaemorrhagiae, and Pomona. Over the past few years, feral swine populations have expanded their geographical range and distribution in the United States with reports in at least 39 of 50 states. Since feral swine serve as reservoirs for serovars that can infect humans, it is important to understand the risk of transmission. In order to learn more about the probability that feral swine shed infectious leptospires, we collected kidneys and paired serum when possible from 677 feral swine in 124 counties of 29 states. These counties had previously been identified as antibody positive for Leptospira interrogans serovars Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae or Pomona. Although exposure to these same six serovars of leptospirosis continued to be high (53% overall) in the counties we sampled, we detected leptospiral DNA in only 3·4% of feral swine kidneys tested. Based on these results, it appears that although feral swine can serve as a source of infection to humans, especially in those who are more likely to encounter them directly such as wildlife biologists, veterinarians, and hunters, the risk may be relatively low. However, further studies to examine the relationship between leptospiral shedding in the urine and kidneys in addition to culturing the organism are recommended in order to better understand the risk associated with feral swine.

Multi-institutional randomized control study of haemolysis in stored red cell units prepared manually or by an automated system.

BACKGROUND: The haemolysis level at the end of storage is a performance parameter for RBC preparations. In the evaluation of new devices or new processes for processing blood, it is relevant to evaluate whether the haemolysis is linked to (1) specific characteristics of the blood donor, or (2) the nature of the blood-processing methodologies. MATERIALS AND METHODS: As part of the validation of a new automated whole blood processing system compared to the current manual methods, randomized, paired crossover studies were conducted evaluating measures of blood component quality, including RBC haemolysis over 42 days of storage. RESULTS: The association between haemolysis and the individual subject was evaluated by modelling haemolysis with independent predictors of treatment (control and test processing) and leucocyte reduction as fixed factors with donor and laboratory as random effects in a mixed-effects ANOVA model. It was found that the day 42 haemolysis values were strongly dependent on the donor subject, with an intraclass correlation coefficient of 0.81. CONCLUSIONS: The data reported in this study suggest a link between the specific whole blood donor and the haemolysis levels observed in red-blood-cell units stored refrigerated for 42 days. Additional research to identify possible donor characteristics associated with haemolysis during storage is warranted.

The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase.

Signal peptides (SPs) target proteins to the secretory pathway and are cleaved from the nascent chain once the translocase in the ER has been engaged. Signal-anchor (SA) sequences also interact transiently with the ER translocase, but are not cleaved and move laterally out of the translocase to become permanent membrane anchors. One obvious difference between SP and SA sequences is the considerably longer hydrophobic regions (h regions) of the latter. To study the interaction between SP/SA sequences and the ER translocase, we have constructed signal sequences with poly-Leu h regions ranging in length from 8 to 29 residues and have characterized their locations within the translocase using both a new assay that measures the minimum number of amino acids needed to span the distance between the COOH-terminal end of the h region and the active site of the oligosaccharyl transferase enzyme and an assay where the efficiency of signal peptidase catalyzed cleavage is measured. Our results suggest that SP and SA sequences are positioned differently in the ER translocase.

Proline-induced disruption of a transmembrane alpha-helix in its natural environment.

alpha-Helix formation in globular proteins has been studied both theoretically and experimentally for decades, while a lack of both high-resolution structures and suitable experimental techniques has hampered the study of helices in membrane proteins. We have developed a new experimental approach, glycosylation mapping, where the active site of the lumenally exposed endoplasmic reticulum enzyme oligosaccharyl transferase is used as a point of reference against which the position of a transmembrane segment in the membrane can be measured. Here, we report an initial analysis of the helix-breaking properties of proline residues inserted in a transmembrane helix. We find that proline residues can break a transmembrane helix, but only when inserted near the end, and only when the helix is sufficiently long. The glycosylation mapping technique may be generally useful for determining the position of transmembrane helices in the membrane.

Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein.

NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologues of these proteins also play an important role in regulation of the Drosophila immune response. Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. We find strong evidence for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appears to be restricted to the IkappaB domain. A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens.

Molecular population genetics of male accessory gland proteins in Drosophila.

Drosophila seminal proteins have an unusually high rate of molecular sequence evolution, suggesting either a high rate of neutral substitution or rapid adaptive evolution. To further quantify patterns of polymorphism and divergence in genes encoding seminal proteins, also called accessory gland proteins (Acp's), we conducted a sequencing survey of 10 Acp genes in samples of Drosophila melanogaster and D. simulans (Acp29AB, Acp32CD, Acp33A, Acp36DE, Acp53Ea, Acp62F, Acp63F, Acp76A, Acp95EF, and Acp98AB). Mean heterozygosity at replacement sites in D. simulans was 0.0074 for Acp genes and 0.0013 for a set of 19 non-Acp genes, and mean melanogaster-simulans divergence at replacement sites was 0.0497 for Acp genes and 0.0107 at non-Acp genes. The elevated divergence of Acp genes is thus accompanied by elevated within-species polymorphism. In addition to the already-reported departures of Acp26A, Acp29AB, and Acp70A from neutrality, our data reject neutrality at Acp29AB and Acp36DE in the direction of excess replacements in interspecific comparisons.

Calmodulin point mutations affect Drosophila development and behavior.

Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations.

Contribution of disulphide bonds to antigenicity of Lep d 2, the major allergen of the dust mite Lepidoglyphus destructor.

To study the contribution of the 3 disulphide bonds in the major allergen Lep d 2 to the antigenic structure, site-directed mutagenesis was performed. Mutants with one or more cysteine residues altered were constructed with a histidine residue tag for purification purposes and expressed as recombinant proteins in E. coli. Seven mutants were analysed: 3 single mutants (Cys 8, Cys 21 and Cys 72), 3 double mutants (Cys 8-117, Cys 21-16 and Cys 72 77) and one mutant with all 6 cysteines altered (6 Cys). The evaluation of IgE reactivity in 10 allergic patients showed that the disulphide bond formed by cysteine 72 and 77 was the single most contributing bond to IgE binding. Mutants with disruption of the Cys 8-117 bond had a lesser reduction in IgE binding, even though this alteration seemed to influence the compact nature of Lep d 2. However, to abolish the IgE reactivity almost completely, all 6 cysteines had to be altered. A monoclonal antibody previously raised against Lepidoglyphus destructor showed a similar binding as human IgE with no reactivity to the Cys 72 77 or the 6 Cys mutant. Using skin prick test we found no reaction to the 6 Cys mutant at the concentrations tested (1-100 microg/ml) in an Lepidoglyphus destructor allergic patient, while the T-cell reactivity was preserved. The 6 Cys mutant of Lep d 2 may, after further evaluation, be a candidate molecule for improved immunotherapy of Lepidoglyphus destructor allergy.

Membrane-protein engineering.

Membrane proteins perform many of the essential functions required for life. They are often the targets of medicinal drugs and have many potential uses in biotechnological processes. Therefore our ability to understand them and manipulate their functions is both important and necessary to enable protein engineers to create 'designer' membrane proteins (that is, proteins designed to have desired properties).

Ala-insertion scanning mutagenesis of the glycophorin A transmembrane helix: a rapid way to map helix-helix interactions in integral membrane proteins.

Alanine insertions into the glycophorin A transmembrane helix are found to disrupt helix-helix dimerization in a way that is fully consistent with earlier saturation mutagenesis data, suggesting that Ala-insertion scanning can be used to rapidly map the approximate location of structurally and/or functionally important segments in transmembrane helices.

Selective cloning of allergens from the skin colonizing yeast Malassezia furfur by phage surface display technology.

The yeast Malassezia furfur, also known as Pityrosporum orbiculare (ovale), is part of the normal microflora of the human skin but has also been associated with different skin diseases including atopic dermatitis. More than 50% of atopic dermatitis patients have positive skin test and specific IgE to M. furfur extracts; however, the pathophysiologic role of these IgE-mediated reactions in the development of the disease remains unknown. The yeast is able to produce a wide panel of IgE-binding proteins, variably recognized by sera of individual patients. In order to assess the contribution of individual components to the disease, highly pure allergen preparations are required. We have cloned M. furfur allergens from a cDNA library displayed on the phage surface, sequenced the inserts and produced recombinant proteins in Escherichia coli. Phage displaying IgE-binding proteins were selectively enriched from the library using IgE from a M. furfur-sensitized atopic dermatitis patient as a ligand. We were able to identify five different inserts coding for IgE-binding polypeptides. Three of the sequenced cDNA encode incomplete gene products with molecular masses of 21.3 kDa (MF 7), 14.4 kDa (MF 8), and 9.7 kDa (MF 9), respectively, having no sequence similarity to known proteins. The other two cDNA encode allergens of 18.2 kDa (Mal f 5) and 17.2 kDa (Mal f 6). Mal f 5 shows significant homology to M. furfur allergens Mal f 2, Mal f 3 and an Aspergillus fumigatus allergen Asp f 3. Mal f 6 has significant homology with cyclophilin. All of the recombinant polypeptides were capable of binding serum IgE from atopic dermatitis patients in immunoblotting experiments. The availability of pure recombinant M. furfur allergens will allow the careful investigation of the role of IgE-binding proteins in atopic dermatitis.

Different conformations of nascent polypeptides during translocation across the ER membrane.

BACKGROUND: In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated alpha-helices) may be possible within the ribosome. RESULTS: We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244). Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro). CONCLUSIONS: The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly alpha-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites.

The DsbA-DsbB system affects the formation of disulfide bonds in periplasmic but not in intramembraneous protein domains.

The DsbA and DsbB proteins of Escherichia coli are involved in facilitating the formation of disulfide bonds in periplasmic proteins. Here, we show that the rate of formation of a disulfide bond in the periplasmic domain of the inner membrane protein leader peptidase is reduced in dsbA and dsbB strains, whereas the rate of formation of a disulfide bond engineered into the membrane embedded domain of the same protein is completely unaffected by these mutations. We conclude that the Dsb proteins do not facilitate the formation of intramembraneous disulfides.

Integrated protective systems for operational acceleration-induced loss of consciousness.

Systems that can protect pilots from not only acceleration-induced loss of consciousness (G-LOC) but also from exposure to chemical, biological, and nuclear weapons are discussed. Hazards such as fire, drowning, and ballistic injury are not considered. Physiological stresses, protection methods, and their impact on G capability at increased altitudes are examined, as are stresses induced by the environment (heat stress, cold stress, and cold water immersion). Sustained and short-duration acceleration effects are described. Requirements for protection against chemical, biological, radiological, and laser weapons and the incorporation of mission-enhancement devices are addressed. Concepts for integration of all of these elements are discussed.

The incidence of AIDS among blacks and Hispanics.

Compared with whites, the acquired immune deficiency syndrome (AIDS) has affected blacks and Hispanics disproportionately. The cumulative incidence (CI) for black men was 2.6, and for Hispanic men 2.5, times the rate for white men. Intravenous (IV) needle use alone does not account for this difference. Not counting IV needle-using cases, the CIs for black and Hispanic men were 1.7 times the CI for white men. Although there were fewer cases in women than men, the white-to-minority disparity was greater for women. The CIs for black and Hispanic women were 12.2 and 8.5 times, respectively, the CI for white women. Prevention programs are urgently needed and should focus on risky behavior (IV needle sharing and receptive anal intercourse), not just risk groups.

Pilot performance of the anti-G straining maneuver: respiratory demands and breathing system effects.

INTRODUCTION: The anti-G straining maneuver (AGSM) is still an important part of pilot protection for G-induced loss of consciousness. The specific requirements for and the effects of breathing systems on the performance of the AGSM are essential elements to designing compatible breathing systems. METHODS: Subject pools of 27 and 34 naval aviators were recruited and used to measure the inhalatory and exhalatory flow requirements for the AGSM and the breathing system effects of mask cavity pressure during AGSM performance on the Naval Air Warfare Center Dynamic Flight Simulator at acceleration levels up to 8 Gz. RESULTS: The mean peak inhalatory flow was 125.5 L.min-1 (n = 135, SD = 42.1) with a maximum value of 274 L.min-1. The mean peak exhalatory flow was 154.4 L.min-1 (n = 135, SD = 49.6) with a maximum value of 308 L.min-1. For the effects of the breathing system on AGSM performance, inhalatory mask cavity pressures were not above 30 mmHg with the majority less than 10 mmHg. Exhalatory mask cavity pressures did not exceed 60 mmHg but predominated in the 20-30 mmHg range. In comparison to accepted guidelines, 67-77% of inhalatory mask cavity pressures were below and 91% of the exhalatory mask cavity pressures were above the Air Standardization and Coordination Committee (ASCC) limit of +/- 14 mmHg. CONCLUSIONS: The difference in the peak inhalatory and exhalatory flows measured during this study and clinically can be attributed to different test conditions and performer techniques. The reduction in inhalatory flow with increasing G is consistent with the increase in breathing difficulty due to the G load and the inflation of the anti-G suit. However, exhalatory mechanics appear unaffected by the G load and the inflation of the anti-G suit. Since 23-33% of the inhalatory mask cavity pressures were above this ASCC limit, improvements in regulator performance are still needed. For exhalatory effects of the breathing system, the main contributor is the mask valve. While no pilot suffered unconsciousness or expressed complaints with the breathing systems used, these exposures were of short duration. The additional work of breathing during a combat engagement may further compromise the pilot's ability to retain consciousness with the AGSM.

The design of arm pressure covers to alleviate pain in high G maneuvers.

HYPOTHESIS: Arm pain has been noted especially when increased coverage anti-G suits are worn in combination with PBG (pressure breathing during G) in high G centrifuge test runs. Arm pain has forced the termination of test runs and has the potential to cause a serious decrement in a pilot's ability to function effectively during high-G flight. Based on the theory that arm pain is caused by fluid pooling in the arm, four new pressure cover systems for the arm were designed and tested. METHODS: Seven test subjects wore each design in a series of runs in a dynamic flight simulator (DFS). Subjects experienced both gradual and rapid onset rate runs with G levels ranging from +3.0 G to +9.0 G. Data were collected on the maximum G's achieved, pain location, and pain intensity. Subjective responses on the comfort and function of each design and overall arm cover design preferences were also obtained via a questionnaire. RESULTS: Analysis of the DFS data, using a two-factor ANOVA (subjects, designs), revealed that each of the new designs provided significantly better protection from arm pain than the control condition (no protection) and that each of the designs significantly improved the maximum G level attained during rapid onset acceleration in the range of +1 G. CONCLUSION: No positive correlation was found between subject ranking of the four new design concepts at the conclusion of the study in terms of the pain reduction they provided and the pain levels reported during DFS testing.

Genetics of alpha-amanitin resistance in a natural population of Drosophila melanogaster.

The genetic basis of variation in resistance to natural toxins is of interest for both ecological and evolutionary genetics. The wide variety of larval resources used by Drosophila, both within and between species, makes flies an excellent system for studying causes and consequences of selection resulting from exposure to natural toxins associated with different resources. In this study we carry out a genetic analysis of alpha-amanitin resistance in a population sample of Drosophila melanogaster. Data from mapping crosses of chromosome III support a role for a naturally occurring polymorphism in a multidrug resistance gene (Mdr65A) in alpha-amanitin resistance. However, there are no amino acid differences between resistant and sensitive chromosomes at Mdr65A. Therefore, if Mdr65A mutants contribute to the difference between alpha-amanitin-resistant and alpha-amanitin-sensitive third chromosome lines, the underlying cause is a gene regulatory mutation.

The pigeon parlance project.

A student laboratory procedure for teaching simple analogs of verbal behavior to pigeons is described. The three kinds of stimulus-response relations are analogs to the topography-based tact, the stimulus-selection-based tact, and receptive language. In the topography-based tact the pigeon exhibits a particular topography (pecking the left foot) when shown a particular nonverbal stimulus (a red plastic ball). In the selection-based tact the pigeon pecks a particular verbal stimulus or lexigram when shown the lexigram. In each case a set of several topographies, nonverbal stimuli, and lexigrams is involved, and food reinforcement is only received if the relation is the correct one. The procedure requires no special apparatus and can be used by students at any level.