An experimental study of the effects of Nosema eurytremae (Microsporida: Nosematidae) on the liver fluke Fasciola hepatica

A microsporidian parasite, Nosema eurytremae, was fed to Lymnaea trunculata infected with Fasciola hepatica. Microsporidian infection of the snail was always light, though spores were present in all tissues but the rediae became heavily infected in the parenchyma. The proportion of infected rediae increased with time and was especially high in ageing infections. Although rediae were only lightly infected when examined before cercarial release began, the infections became progressively heavier, so that towards the end of the life of the snails a high proportion of rediae were totally destroyed and contained no cercariae. Cercarial output was significantly depressed over a long period of active shedding by snails which had been hyperinfected with 1 x 106 spores and some of the liberated cercariae were themselves infected.     

'Pals'. A medical student public service program.

We designed a public service and educational program to aid children and families coping with chronic illness and to augment medical student education. Medical students developed relationships with chronically ill children and families based on the Big Brother-Big Sister program model. In addition, students attended bimonthly seminars on childhood chronic illness and family dynamics. Medical students learned about the psychosocial aspects of illness through these relationships and reported that the program contributed to their sense of worth as caregivers. By fostering students'' innate altruism, medical schools may succeed in cultivating caring and humanism in their student physicians. We propose a model that encourages medical students to relate personally with patients and their families. A program such as this has the potential to nurture compassion in medical students, contribute to medical education, and provide support to patients and families.     

Reflex regulation of airway smooth muscle tone.

Autonomic nerves in most mammalian species mediate both contractions and relaxations of airway smooth muscle. Cholinergic-parasympathetic nerves mediate contractions, whereas adrenergic-sympathetic and/or noncholinergic parasympathetic nerves mediate relaxations. Sympathetic-adrenergic innervation of human airway smooth muscle is sparse or nonexistent based on histological analyses and plays little or no role in regulating airway caliber. Rather, in humans and in many other species, postganglionic noncholinergic parasympathetic nerves provide the only relaxant innervation of airway smooth muscle. These noncholinergic nerves are anatomically and physiologically distinct from the postganglionic cholinergic parasympathetic nerves and differentially regulated by reflexes. Although bronchopulmonary vagal afferent nerves provide the primary afferent input regulating airway autonomic nerve activity, extrapulmonary afferent nerves, both vagal and nonvagal, can also reflexively regulate autonomic tone in airway smooth muscle. Reflexes result in either an enhanced activity in one or more of the autonomic efferent pathways, or a withdrawal of baseline cholinergic tone. These parallel excitatory and inhibitory afferent and efferent pathways add complexity to autonomic control of airway caliber. Dysfunction or dysregulation of these afferent and efferent nerves likely contributes to the pathogenesis of obstructive airways diseases and may account for the pulmonary symptoms associated with extrapulmonary disorders, including gastroesophageal reflux disease, cardiovascular disease, and rhinosinusitis.     

Bicyclic monoterpene diols stimulate release of nitric oxide from skin cells, increase microcirculation, and elevate skin temperature.

Bicyclic monoterpene diols (BMTd) stimulate nitric oxide synthesis in melanoma and neuronal cells, representing cell types arising from embryonic neural crest tissue. This study shows that an equimolar mixture of the BMTd's 2,3-cis/exo-pinanediol and 2,3-cis/exo-camphanediol stimulates nitric oxide synthesis in epithelial cells of the skin, specifically normal human epidermal keratinocytes (NHEK) and normal human microvascular endothelial cells (HMVEC). A 1 mM mixture increased nitric oxide 3-fold in HMVEC in the first 24 h after treatment, and a 2 mM mixture produced an equivalent increase in NHEK. We hypothesized that an increase in nitric oxide in skin would lead to an increase in microcirculation, thereby increasing skin temperature. We found that twice daily application of 1mM BMTd lotion significantly increased arm skin temperature by 0.5 degrees C in 14 days compared to placebo, while a 2 mM mixture significantly increased skin temperature by 0.3 degrees C in 7 days (P < or = 0.05; ANOVA). A single application of a 2 mM BMTd mixture applied 30 min before a 30 min cold challenge (6 degrees C), maintained facial skin temperature 1.4 degrees C above untreated control sites (P < or = 0.05; ANOVA). We also tested whether BMTd treatment would benefit people with dark circles under their eyes. Twenty-six panelists with dark undereye circles completed 2-week, twice daily application of a lotion containing the 1mM mixture to one eye while the other eye was untreated. Seven of 26 subjects showed a reduction of darkness of undereye circles (P < or = 0.05; paired t test). Application of 2 mM BMTd lotion to lips resulted in a significant increase in their redness, as measured by the erythema index (P < or = 0.05; ANOVA). These results show that a mixture of BMTd's increases nitric oxide, and application to skin increases microcirculation and skin temperature.     

Structural Basis for Cul3 Protein Assembly with the BTB-Kelch Family of E3 Ubiquitin Ligases

Cullin-RING ligases are multisubunit E3 ubiquitin ligases that recruit substrate-specific adaptors to catalyze protein ubiquitylation. Cul3-based Cullin-RING ligases are uniquely associated with BTB adaptors that incorporate homodimerization, Cul3 assembly, and substrate recognition into a single multidomain protein, of which the best known are BTB-BACK-Kelch domain proteins, including KEAP1. Cul3 assembly requires a BTB protein “3-box” motif, analogous to the F-box and SOCS box motifs of other Cullin-based E3s. To define the molecular basis for this assembly and the overall architecture of the E3, we determined the crystal structures of the BTB-BACK domains of KLHL11 both alone and in complex with Cul3, along with the Kelch domain structures of KLHL2 (Mayven), KLHL7, KLHL12, and KBTBD5. We show that Cul3 interaction is dependent on a unique N-terminal extension sequence that packs against the 3-box in a hydrophobic groove centrally located between the BTB and BACK domains. Deletion of this N-terminal region results in a 30-fold loss in affinity. The presented data offer a model for the quaternary assembly of this E3 class that supports the bivalent capture of Nrf2 and reveals potential new sites for E3 inhibitor design.     

Encoding of the cough reflex in anesthetized guinea pigs

We have previously described the physiological and morphological properties of the cough receptors and their sites of termination in the airways and centrally in the nucleus tractus solitarius (nTS). In the present study, we have addressed the hypothesis that the primary central synapses of the cough receptors subserve an essential role in the encoding of cough. We found that cough requires sustained, high-frequency (≥8-Hz) afferent nerve activation. We also found evidence for processes that both facilitate (summation, sensitization) and inhibit the initiation of cough. Sensitization of cough occurs with repetitive subthreshold activation of the cough receptors or by coincident activation of C-fibers and/or nTS neurokinin receptor activation. Desensitization of cough evoked by repetitive and/or continuous afferent nerve activation has a rapid onset (<60 s) and does not differentiate between tussive stimuli, suggesting a central nervous system-dependent process. The cough reflex can also be actively inhibited upon activation of other airway afferent nerve subtypes, including slowly adapting receptors and pulmonary C-fibers. The sensitization and desensitization of cough are likely attributable to the prominent, primary, and unique role of N-methyl-d-aspartate receptor-dependent signaling at the central synapses of the cough receptors. These attributes may have direct relevance to the presentation of cough in disease and for the effectiveness of antitussive therapies.     

Molecular dynamics, crystallography and mutagenesis studies on the substrate gating mechanism of prolyl oligopeptidase

Altered prolyl oligopeptidase (PREP) activity is found in many common neurological and other genetic disorders, and in some cases PREP inhibition may be a promising treatment. The active site of PREP resides in an internal cavity; in addition to the direct interaction between active site and substrate or inhibitor, the pathway to reach the active site (the gating mechanism) must be understood for more rational inhibitor design and understanding PREP function. The gating mechanism of PREP has been investigated through molecular dynamics (MD) simulation combined with crystallographic and mutagenesis studies. The MD results indicate the inter-domain loop structure, comprised of 3 loops at residues, 189-209 (loop A), 577-608 (loop B), and 636-646 (loop C) (porcine PREP numbering), are important components of the gating mechanism. The results from enzyme kinetics of PREP variants also support this hypothesis: When loop A is (1) locked to loop B through a disulphide bridge, all enzyme activity is halted, (2) nicked, enzyme activity is increased, and (3) removed, enzyme activity is only reduced. Limited proteolysis study also supports the hypothesis of a loop A driven gating mechanism. The MD results show a stable network of H-bonds that hold the two protein domains together. Crystallographic study indicates that a set of known PREP inhibitors inhabit a common binding conformation, and this H-bond network is not significantly altered. Thus the domain separation, seen to occur in lower taxa, is not involved in the gating mechanism for mammalian PREP. In two of the MD simulations we observed a conformational change that involved the breaking of the H-bond network holding loops A and B together. We also found that this network was more stable when the active site was occupied, thus decreasing the likelihood of this transition.