Characterisation of N-linked protein glycosylation in the bacterial pathogen Campylobacter hepaticus.

Campylobacter hepaticus is an important pathogen which causes Spotty Liver Disease (SLD) in layer chickens. SLD results in an increase in mortality and a significant decrease in egg production and therefore is an important economic concern of the global poultry industry. The human pathogen Campylobacter jejuni encodes an N-linked glycosylation system that plays fundamental roles in host colonization and pathogenicity. While N-linked glycosylation has been extensively studied in C. jejuni and is now known to occur in a range of Campylobacter species, little is known about C. hepaticus glycosylation. In this study glycoproteomic analysis was used to confirm the functionality of the C. hepaticus N-glycosylation system. It was shown that C. hepaticus HV10T modifies > 35 proteins with an N-linked heptasaccharide glycan. C. hepaticus shares highly conserved glycoproteins with C. jejuni that are involved in host colonisation and also possesses unique glycoproteins which may contribute to its ability to survive in challenging host environments. C. hepaticus N-glycosylation may function as an important virulence factor, providing an opportunity to investigate and develop a better understanding the system's role in poultry infection.

A Toxic Synergy between Aluminium and Amyloid Beta in Yeast.

Alzheimer's disease (AD), the most prevalent, age-related, neurodegenerative disease, is associated with the accumulation of amyloid beta (Aß) and oxidative stress. However, the sporadic nature of late-onset AD has suggested that other factors, such as aluminium may be involved. Aluminium (Al3+) is the most ubiquitous neurotoxic metal on earth, extensively bioavailable to humans. Despite this, the link between Al3+ and AD has been debated for decades and remains controversial. Using Saccharomyces cerevisiae as a model organism expressing Aß42, this study aimed to examine the mechanisms of Al3+ toxicity and its interactions with Aß42. S. cerevisiae cells producing Aß42 treated with varying concentrations of Al3+ were examined for cell viability, growth inhibition, and production of reactive oxygen species (ROS). Al3+ caused a significant reduction in cell viability: cell death in yeast producing green fluorescent protein tagged with Aß42 (GFP-Aß42) was significantly higher than in cells producing green fluorescent protein (GFP) alone. Additionally, Al3+ greatly inhibited the fermentative growth of yeast producing GFP-Aß42, which was enhanced by ferric iron (Fe3+), while there was negligible growth inhibition of GFP cells. Al3+- induced ROS levels in yeast expressing native Aß42 were significantly higher than in empty vector controls. These findings demonstrate Al3+ has a direct, detrimental toxic synergy with Aß42 that can be influenced by Fe3+, causing increased oxidative stress. Thus, Al3+ should be considered as an important factor, alongside the known characteristic hallmarks of AD, in the development and aetiology of the disease.

Regulation of hypothalamic corticotropin-releasing hormone neurone excitability by oxytocin.

Oxytocin (OT) is a neuropeptide that exerts multiple actions throughout the brain and periphery. Within the brain, OT regulates diverse neural populations, including neural networks controlling responses to stress. Local release of OT within the paraventricular nucleus (PVN) of the hypothalamus has been suggested to regulate stress responses by modulating the excitability of neighbouring corticotropin-releasing hormone (CRH) neurones. However, the mechanisms by which OT regulates CRH neurone excitability are unclear. In the present study, we investigated the morphological relationship between OT and CRH neurones and determined the effects of OT on CRH neurone excitability. Morphological analysis revealed that the processes of OT and CRH neurones were highly intermingled within the PVN, possibly allowing for local cell-to-cell cross-talk. Whole-cell patch-clamp recordings from CRH neurones were used to study the impact of OT on postsynaptic excitability and synaptic innervation. Bath-applied OT did not alter CRH neurone holding current, spiking output or any action potential parameters. Recordings of evoked excitatory and inhibitory postsynaptic currents (EPSCs/IPSCs) revealed no net effect of OT on current amplitude; however, subgroups of CRH neurones appeared to respond differentially to OT. Analysis of spontaneous EPSC events uncovered a significant reduction in spontaneous EPSC frequency but no change in spontaneous EPSC amplitude in response to OT. Together, these data demonstrate that OT exerts a subtle modulation of synaptic transmission onto CRH neurones providing one potential mechanism by which OT could suppress CRH neurone excitability and stress axis activity.

Stress Physiology in Infancy and Early Childhood: Cortisol Flexibility, Attunement and Coordination.

Research on stress physiology in infancy has assumed increasing importance due to its lifelong implications. In this review, we focus on measurement of hypothalamic-pituitary-adrenal (HPA) function, in particular, and on complementary autonomic processes. We suggest that the measure of HPA function has been overly exclusive, focusing on individual reactivity to single, pragmatically selected laboratory challenges. We advocate use of multiple, strategically chosen challenges and within-subject designs. By administering one challenge that typically does not provoke reactivity and another that does, it is possible to represent allostatic load in terms of "flexibility," the capacity to titrate response to challenge. We also recommend assessing infant reactivity in the context of the primary caregiver's physiological function. Infant-mother "attunement" is central to developmental psychology, permeating diverse developmental domains with varied consequences. A review of adrenocortical attunement suggests that attunement is a reliable process, manifest across varied populations. However, attunement appears stronger in the context of more highly stressful circumstances, such that administration of multiple, selected challenges may help evaluate the degree to which individuals titrate attunement to challenge and determine the correlates of this differential attunement. Finally, we advocate studying the "coordination" of HPA function with other aspects of stress physiology and variation in the degree of this coordination. The use of multiple stressors is important here because each stress system is differentially sensitive to different types of challenge. Therefore, use of single stressors in between-subject designs impedes full recognition of the role played by each system. Overall, we recommend measure of flexibility, attunement, and coordination in the context of multiple challenges to capture allostasis in environmental and physiological context. The simultaneous use of such inclusive and integrative metrics may yield more reliable findings than has hitherto been the case. The interrelation of these metrics can be understood in the context of the adaptive calibration model..

Preferential depletion of gut CD4-expressing iNKT cells contributes to systemic immune activation in HIV-1 infection.

Chronic inappropriate immune activation is the central defect-driving loss of CD4(+) T helper cells and progression to AIDS in persons with HIV-1 infection, but the mechanisms remain controversial. We examined key regulatory invariant receptor natural killer T (iNKT) cells in the gut, the largest reservoir of lymphocytes and a key arena of HIV-1 pathogenesis. In healthy control persons, the anti-inflammatory CD4(+) iNKT-cell subset predominated over the pro-inflammatory CD4(-) iNKT-cell subset in the gut, but not in the blood, compartment. HIV-1 infection resulted in a preferential loss of this anti-inflammatory CD4(+) iNKT-cell subset within the gut. The degree of loss of the CD4(+) iNKT-cell subset in the gut, but not in the blood, correlated to the systemic immune activation and exhaustion that have been linked to disease progression. These results suggest a potentially important contribution of gut iNKT-cell imbalance in determining the systemic immune activation that is the hallmark of HIV-1 pathogenesis.

Drug abuse treatment beyond prison walls.

The period surrounding release from prison is a critical time for parolees, bearing the potential for a drug-free and crime-free life in the community but also high risks for recidivism and relapse to drugs. The authors describe two projects. The first illustrates the use of a formal Delphi process to elicit and combine the expertise of treatment providers, researchers, corrections personnel, and other stakeholders in a set of statewide guidelines for facilitating re-entry. The second project is a six-session intervention to enable women to protect themselves against acquiring or transmitting HIV in their intimate relationships.

Toxic effects of Streptomyces griseus spores and exudate on gill pathology of freshwater fish.

Many unexplained fish-kills in British waters are considered microbial in origin and a large proportion of field sites contains elevated concentrations of filamentous actinobacteria. The present study has shown that a strain of Streptomyces griseus, isolated from field sites, elicits pathological changes to the gills of fish under laboratory conditions which mirror those found in situ. These changes include hyperplasia leading to fusion of the secondary lamellae and loss of microridging on the filamental epithelium of the primary lamellae. Juveniles of up to six fish species were exposed to spore suspensions or exudate of S. griseus in the range of 1 x 10(2)-1 x 10(6)spores ml(-1) for up to 96 h. The exudate was more potent than the spores and there was a positive correlation between exudate concentration and the rate and extent of fish gill pathology with bream and rainbow trout being more sensitive than carp, tench and roach. The results are discussed in the context of recognising and managing potential fish mortalities caused by microbial toxins.

Microfabricated silicon leak for sampling planetary atmospheres with a mass spectrometer.

A microfabricated silicon mass spectrometer inlet leak has been designed, fabricated, and tested. This leak achieves a much lower conductance in a smaller volume than is possible with commonly available metal or glass capillary tubing. It will also be shown that it is possible to integrate significant additional functionality, such as inlet heaters and valves, into a silicon microleak with very little additional mass. The fabricated leak is compatible with high temperature (up to 500 degrees C) and high pressure (up to 100 bars) conditions, as would be encountered on a Venus atmospheric probe. These leaks behave in reasonable agreement with their theoretically calculated conductance, although this differs between devices and from the predicted value by as much as a factor of 2. This variation is believed to be the result of nonuniformity in the silicon etching process which is characterized in this work. Future versions of this device can compensate for characterized process variations in order to produce devices in closer agreement with designed conductance values. The integration of an inlet heater into the leak device has also been demonstrated in this work.

[Delleman syndrome: anesthetic considerations]. / Syndrome de Delleman: prise en charge anesthésique.

We herein report a case of a 5-year-old patient with Delleman Syndrome, a rare congenital disorder affecting the eyes, skin, and central nervous system, who underwent general anesthesia for conjuctivoplasty. This is only the second report of the anesthetic management of a patient with this condition. We attempt to summarize some of the anesthetic implications of this syndrome.

Measurement of the Michel parameter rho in muon decay.

The TWIST Collaboration has measured the Michel parameter rho in normal muon decay, mu(+)--> e(+)nu(e)nu (mu). In the standard model, rho = 3/4. Deviations from this value imply mixing of left- and right-handed muon and electron couplings. We find rho=0.750 80+/-0.000 32(stat) +/- 0.000 97(syst) +/- 0.000 23, where the last uncertainty represents the dependence of rho on the Michel parameter eta. This result sets new limits on the W(L)-W(R) mixing angle in left-right symmetric models.

Effects of asthma on cell components in peripheral blood among smokers and non-smokers.

BACKGROUND: Eosinophils play a central role in asthma, but the interplay of the effects of smoking, eosinophils and asthma remains unclear. OBJECTIVE: The primary objective of our study was to investigate the extent to which smoking modifies the effect of asthma on circulating eosinophils, CD4+ and CD8+ T cell counts. METHODS: Data were collected semiannually between 1987 and 1994 from HIV-negative participants in the Multicenter AIDS Cohort Study. Asthma was defined by a questionnaire at baseline as a self-report of diagnosed asthma. A total of 1420 blood samples from 197 asthmatics and 15 822 from 1997 non-asthmatics were collected. RESULTS: Eosinophil levels were higher in asthmatics (28% of asthmatics had eosinophils >/=4% and 16% of non-asthmatics) regardless of smoking history, but smoking modified the association between eosinophils and asthma. Namely, the odds ratios for eosinophils being >/=4% in asthmatics to non-asthmatics decreased from 2.7 (95% CI: 2.0, 3.6) in never, to 2.1 (1.4, 3.1) in former, and to 1.5 (0.9, 2.3) in current smokers. Cross-sectional and longitudinal analyses coherently showed that smoking increased eosinophils in non-asthmatics, but the converse was true for asthmatics. In contrast, no differences in peripheral blood T cell counts between asthmatics and non-asthmatics were observed. CONCLUSION: Under the established link between increased eosinophils and asthma, these data indicate that smoking modified this relationship. This finding suggests that smoking plays a different immunological role in asthmatics and non-asthmatics.

Genotyping TAP2 variants in North American Caucasians, Brazilians, and Africans.

The protein forms of transporter associated with antigen processing, subunit 2 (TAP2), differ either by amino acid substitutions (Thr374Ala, Ile379Val, Ile467Val, Thr565Ala, Val577Met, Cys651Arg, and Ala665Thr) or by a truncation (Gln687Stop) of 17 amino acid residues at the C-terminus. Nonsynonymous single nucleotide polymorphisms (N-SNPs) causing these amino acid variations except 577Val were detected in genomic DNA samples from North American Caucasians (n = 76), Brazilians (n = 148), Rwandans (n = 285), and Zambians (n = 117). Exclusive (100%) and nearly exclusive (>95%) linkage disequilibrium was seen with a number of N-SNPs. The average heterozygosity at any given dimorphic site ranged from 7.3% to 44.6%, and at least four N-SNPs showed clear population specificity. N-SNP combinations alone led to the identification of 16 relatively common alleles, which appeared to form at least three lineages. Further analyses of 101 cDNA samples from Brazilians detected nine expressed TAP2 alleles, four of which matched the official assignments. Genetic complexity at the TAP2 locus was further enhanced by two out of five synonymous SNPs (S-SNPs), especially the GGT386GGG (Gly) that had similar heterozygosity rates in Caucasians (28.9%), Rwandans (33.3%), and Zambians (33.3%). Overall, distribution of both synonymous and nonsynonymous SNPs in the various ethnic groups examined here conformed well to the Hardy-Weinberg equilibrium, and between 57.9% and 77.0% of subjects in each ethnic group were heterozygous with two TAP2 alleles predicted to differ by at least one amino acid residue. Such complexity of TAP2 polymorphisms, in the form of SNPs as well as alleles, is likely to complicate the analyses of disease associations and haplotype structures in the HLA class II region.

Comparison of cell cycle arrest, transactivation, and apoptosis induced by the simian immunodeficiency virus SIVagm and human immunodeficiency virus type 1 vpr genes.

All primate lentiviruses known to date contain one or two open reading frames with homology to the human immunodeficiency virus type 1 (HIV-1) vpr gene. HIV-1 vpr encodes a 96-amino-acid protein with multiple functions in the viral life cycle. These functions include modulation of the viral replication kinetics, transactivation of the long terminal repeat, participation in the nuclear import of preintegration complexes, induction of G2 arrest, and induction of apoptosis. The simian immunodeficiency virus (SIV) that infects African green monkeys (SIVagm) contains a vpr homologue, which encodes a 118-amino-acid protein. SIVagm vpr is structurally and functionally related to HIV-1 vpr. The present study focuses on how three specific functions (transactivation, induction of G2 arrest, and induction of apoptosis) are related to one another at a functional level, for HIV-1 and SIVagm vpr. While our study supports previous reports demonstrating a causal relationship between induction of G2 arrest and transactivation for HIV-1 vpr, we demonstrate that the same is not true for SIVagm vpr. Transactivation by SIVagm vpr is independent of cell cycle perturbation. In addition, we show that induction of G2 arrest is necessary for the induction of apoptosis by HIV-1 vpr but that the induction of apoptosis by SIVagm vpr is cell cycle independent. Finally, while SIVagm vpr retains its transactivation function in human cells, it is unable to induce G2 arrest or apoptosis in such cells, suggesting that the cytopathic effects of SIVagm vpr are species specific. Taken together, our results suggest that while the multiple functions of vpr are conserved between HIV-1 and SIVagm, the mechanisms leading to the execution of such functions are divergent.

Descriptions of the mature spermatozoa of the lizards Crotaphytus bicinctores, Gambelia wislizenii (Crotaphytidae), and Anolis carolinensis (Polychrotidae) (Reptilia, Squamata, Iguania).

The spermatozoa of Crotaphytus bicinctores and Gambelia wislizenii (Crotaphytidae), and Anolis carolinensis (Polychrotidae) exhibit the squamate autapomorphies of a single perforatorium extending anteriorly from the apical tip of the paracrystalline subacrosomal cone, the presence of an epinuclear electron-lucent region, and extension of the fibrous sheath into the midpiece. Crotaphytid sperm differ from those of polychrotids in several respects, including: the structure of the perforatorium, the size of the epinuclear electron-lucent region, aspects of the acrosome complex, the arrangement and structure of intermitochondrial dense bodies, and in the distance the fibrous sheath extends into the midpiece. The sperm of C. bicinctores, G. wislizenii, and A. carolinensis are most similar to those of the agamids and phrynosomatids examined to date. No spermatozoal autapomorphies for Crotaphytidae or Polychrotidae were found. The condition of having the intermitochondrial dense bodies arranged in regular incomplete rings is tentatively defined as a synapomorphy of Iguania (although modified in Chamaeleonidae). Spermatozoal ultrastructure offers no characters that justify the separation of Iguanidae (sensu lato) into several separate families.

Simultaneous flow cytometric measurement of viability and lymphocyte subset proliferation.

Combined analysis of DNA content and immunofluorescence on single cells by flow cytometry provides information on the proliferative response of cellular sub-populations in mixed cell preparations. However, the presence of considerable numbers of dead (nonviable) cells impairs accurate flow cytometric data analysis, mainly, because dead cells can bind antibodies non-specifically and show alterations in their DNA staining profiles. We developed a rapid method for identification of dead cells by fluorescence in cell preparations that are stained simultaneously for two-color immunofluorescence and DNA content. Cells are stained with 7-aminoactinomycin D (7-AAD) for dead cell discrimination and with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE)-labeled monoclonal antibodies (mAb) for cell surface immunofluorescence. Diffusion of 7-AAD from stained, dead cells into unstained, live cells after cell permeabilization is blocked by the addition of its non-fluorescent analogue actinomycin D (AD). DNA is stained with red-excitable TO-PRO-3 iodide (TP3) which has an emission spectrum that can be effectively separated from the emissions of FITC, PE, and 7-AAD. TP3 staining is performed in the presence of ribonuclease A (RNAse) in phosphate-citrate buffer containing saponin (PCBS) at low pH. FITC fluorescence is sensitive to acid pH; therefore, PCBS is replaced after DNA staining with 1x PBS at pH 7.2 containing saponin to permit accurate detection of FITC immunofluorescence on the flow cytometer. We apply this method to the analysis of differential proliferation of lymphocyte subsets in cultures of human peripheral blood mononuclear cells (PBMC) with low viability.

Age-related effects of blocked and random practice schedules on learning a new technology.

Computer technology is pervasive in today's society. Issues of training must be investigated to ensure that older individuals are capable of interacting with such technology. In the present research a simulated automatic teller machine (ATM) served as a prototypical technology for which issues of training and transfer could be investigated. The focus of the study was on the potential benefits of a random practice schedule (wherein trial types are intermixed) relative to a blocked practice schedule (wherein trial types are grouped together). Both younger and older adults benefited from random practice for the acquisition of the ability to perform transactions on an ATM. Moreover, random practice was beneficial for both age groups in the transfer of learning to novel tasks on a novel ATM. These data have general implications for theories of training and specific implications for the development of training protocols for older adults and new technologies.

Morphology of the spermatozoa of the iguanian lizards Uta stansburiana and Urosaurus ornatus (Squamata, Phrynosomatidae).

The spermatozoa of Uta stansburiana and Urosaurus ornatus show the following squamate autapomorphies: a single perforatorium extending anteriorly from the apical tip of the paracrystalline subacrosomal cone; the presence of an epinuclear electron lucent region; intermitochondrial dense bodies; and the fibrous sheath extending into the midpiece. The acrosome vesicle is flattened and concentrically zoned apically; basally it overlies a subacrosomal cone which invests the nuclear rostrum. A stopper-like perforatorial base plate, rounded nuclear shoulders and a basal nuclear fossa are present. The proximal centriole contains a density within its centre for approximately one half its length and lies at approximately 80 degrees to the distal centriole. The two central singlets of the axoneme extend into the short distal centriole. A peripheral dense fibre is associated with each of the nine triplets of the distal centriole, and the fibre continues posteriorly with each of the nine doublets of the axoneme. A central fibre is associated with the two central singlets. All fibres are absent or vestigial at the level of the annulus. Mitochondria are short sinuous with a maximum of eight seen in transverse section. Uta and Urosaurus sperm differ from each other in their arrangement of intermitochondrial dense bodies in two ways: 1) longitudinally, Uta has five incomplete 'rings' of dense bodies, whereas Urosaurus has only four such rings; 2) in cross section, each individual 'ring' of Uta may contain up to four irregularly spaced dense bodies, whereas Urosaurus contains a maximum of only two dense bodies. The sperm of Uta and Urosaurus show strong similarities to those of the agamids and polychrotids. No spermatozoal autapomorphies for the Phrynosomatidae were found.

Phylogeny of the monopisthocotylea and Polyopisthocotylea (Platyhelminthes) inferred from 28S rDNA sequences.

This study focuses on the phylogenetic relationships within the Polyopisthocotylea and Monopisthocotylea, two groups that are often grouped within the monogeneans, a group of disputed paraphyly. Phylogenetic analyses were conducted with multiple outgroups chosen according to two hypotheses, a paraphyletic Monogenea or a monophyletic Monogenea, and with three methods, namely maximum parsimony, neighbour joining and maximum likelihood. Sequences used were from the partial domain C1, full domain D1, and partial domain C2 (550 nucleotides, 209 unambiguously aligned sites) from the 28S ribosomal RNA gene for 16 species of monopisthocotyleans, 26 polyopisthocotyleans including six polystomatids, and other Platyhelminthes (61 species in total, 27 new sequences). Results were similar with outgroups corresponding to the two hypotheses. Within the Monopisthocotylea, relationships were: ¿[(Udonella, capsalids), monocotylids], (diplectanids, ancyrocephalids)¿; each of these families was found to be monophyletic and their monophyly was supported by high bootstrap values in neighbour joining and maximum parsimony. Within the Polyopisthocotylea, the polystomatids were the sister-group of all others. Among the latter, Hexabothrium, parasite of chondrichthyans, was the most basal, and the mazocraeids, mainly parasites of clupeomorph teleosts, were the sister-groups of all other studied polyopisthocotyleans, these, mainly parasites of euteleosts, being polytomous.