Selective modulation of task performance by octopamine in honey bee (<Emphasis Type="Italic">Apis mellifera</Emphasis>) division of labour

Octopamine treatment has previously been shown to increase honey bee foraging behaviour. We determined the effects of octopamine on other tasks to learn how octopamine affects division of labour in honey bee colonies. Octopamine treatment did not increase the rate of corpse removal from the hive, suggesting that elevated brain levels of octopamine do not act to increase the performance of all flight-related tasks. Octopamine treatment also did not increase attendance in the queen's retinue, suggesting that elevated brain levels of octopamine do not act to increase responsiveness to all olfactory stimuli. Consistent with these findings, octopamine treatment enhanced the foraging response to brood pheromone but not the cell capping response, a component of brood care. These results demonstrate a relatively specific form of neuromodulation by octopamine in the regulation of division of labour in honey bee colonies.     

Printing of protein microarrays via a capillary-free fluid jetting mechanism

Current proteomics experiments rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the requirements needed for successful spotting of proteins to perform high-throughput, array-based proteomic profiling. Biological laser printing (BioLP) is a spotting technology that does not rely on solid pins, quill pins, or capillary-based fluidics. The non-contact mechanism of BioLP utilizes a focused laser pulse to transfer protein solutions, thereby eliminating the potential for orifice clogging, air bubbles, and unnecessary volume loss potentially encountered in commercially available spotting technologies. The speed and spot-to-spot reproducibility of BioLP is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microm and approximately 500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. Arraying a solution of BSA with subsequent immunodetection demonstrates the reproducible spotting of protein in an array format with CVs of <3%. Printing of the enzyme alkaline phosphatase followed by a positive reaction with a colorimetric substrate demonstrates that functional protein can be spotted using this laser-based printer.     

Leishmania braziliensis: a novel mechanism in the lipophosphoglycan regulation during metacyclogenesis

During metacyclogenesis of Leishmania in its sand fly vector, the parasite differentiates from a noninfective, procyclic form to an infective, metacyclic form, a process characterised by morphological changes of the parasite and also biochemical transformations in its major surface lipophosphoglycan (LPG). This lipid-anchored polysaccharide is polymorphic among species with variations in sugars that branch off the conserved Gal(beta1,4)Man(alpha1)-PO4 backbone of repeat units and the oligosaccharide cap. Lipophosphoglycan has been implicated as an adhesion molecule that mediates the interaction with the midgut epithelium of the sand fly in the subgenus Leishmania. This paper describes the LPG structure for the first time in a species from the subgenus Viannia, Leishmania (Viannia) braziliensis. The LPG from the procyclic form of L. braziliensis was found to lack side chain sugar substitutions. In contrast to other species from the subgenus Leishmania, metacyclic forms of L. braziliensis makes less LPG and add 1-2 (beta1-3) glucose residues that branch off the disaccharide-phosphate repeat units of LPG. Thus, this represents a novel mechanism in the regulation of LPG structure during metacyclogenesis.     

Nanoscale enzyme inhibitors: Fullerenes inhibit carbonic anhydrase by occluding the active site entrance

We investigated a series of derivatized fullerenes possessing alcohol, amine, and amino acid pendant groups as inhibitors of the zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1). We discovered that fullerenes bind CAs with submicromolar—low micromolar affinity, despite the fact that these compounds do not possess moieties normally associated with CA inhibitors such as the sulfonamides and their isosteres, or the coumarins. The 13 different mammalian CA isoforms showed a diverse inhibition profile with these compounds. By means of computational methods we assessed the inhibition mechanism as being due to occlusion of the active site entrance by means of the fullerene cage (possessing dimension of the same order of magnitude as the opening of the enzyme cavity, of 1 nm). The pendant moieties to the fullerene cage make interactions with amino acid residues from the active site, among which His64, His94, His96, Val121, and Thr200. Fullerenes thus represent a totally new class of nanoscale CA inhibitors which may show applications for targeting physiologically relevant isoforms, such as the dominant CA II and the tumor-associated CA IX.     

Glycosylation of PrPC Determines Timing of Neuroinvasion and Targeting in the Brain following Transmissible Spongiform Encephalopathy Infection by a Peripheral Route

Transmissible spongiform encephalopathy (TSE) infectivity naturally spreads from site of entry in the periphery to the central nervous system where pathological lesions are formed. Several routes and cells within the host have been identified as important for facilitating the infectious process. Expression of the glycoprotein cellular PrP (PrP^C) is considered a key factor for replication of infectivity in the central nervous system (CNS) and its transport to the brain, and it has been suggested that the infectious agent propagates from cell to cell via a domino-like effect. However, precisely how this is achieved and what involvement the different glycoforms of PrP have in these processes remain to be determined. To address this issue, we have used our unique models of gene-targeted transgenic mice expressing different glycosylated forms of PrP. Two TSE strains were inoculated intraperitoneally into these mice to assess the contribution of diglycosylated, monoglycosylated, and unglycosylated PrP in spreading of infectivity to the brain. This study demonstrates that glycosylation of host PrP has a profound effect in determining the outcome of disease. Lack of diglycosylated PrP slowed or prevented disease onset after peripheral challenge, suggesting an important role for fully glycosylated PrP in either the replication of the infectious agent in the periphery or its transport to the CNS. Moreover, mice expressing unglycosylated PrP did not develop clinical disease, and mice expressing monoglycosylated PrP showed strikingly different neuropathologic features compared to those expressing diglycosylated PrP. This demonstrates that targeting in the brain following peripheral inoculation is profoundly influenced by the glycosylation status of host PrP.Transmissible spongiform encephalopathies (TSE) or prion diseases are a group of fatal neurodegenerative diseases which include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathies (BSE) in cattle, and chronic wasting disease (CWD) in deer and elk (30). These diseases can be sporadic, familial, or acquired by infection, and the common hallmark is a distinct pathology in the central nervous system (CNS) characterized by neuronal loss, spongiform degeneration, and gliosis (38, 46).Expression of the host-encoded cellular PrP (PrP^C) is fundamental for the onset of disease since PrP-deficient mice are refractory to TSE infection (11, 31). PrP^C is a glycoprotein with two consensus sites for attachment of N-linked glycans (at codons 180 and 196 in the mouse) which are variably occupied, producing di-, mono-, and unglycosylated PrP (43). The diversity in glycosylation, combined with the complexity of added sugars, results in a large number of glycosylated forms of PrP (41). A central event associated with TSE infection is the conformational conversion of PrP^C into an abnormal protease-resistant form, PrP^Sc (39). PrP^Sc is deposited in brain and, in some but not all cases, in peripheral organs of individuals affected by TSE (21).Although the pathology associated with TSE is found in the brain, the periphery is the most natural route of acquiring infection. Evidence suggests that oral transmission via contaminated food is linked with transmission of BSE to humans, resulting in variant CJD (vCJD) (10, 47), and blood transfusion has been identified as a probable route of human-to-human transmission of vCJD (23, 27, 36). Moreover, parenteral administration of contaminated human tissue-derived therapeutics has been shown to facilitate iatrogenic spread of these diseases (8, 46). It is therefore important to understand the mechanisms that allow the infectious agent to propagate in the periphery and be transported to the CNS prior to the onset of neurodegeneration in the brain.Many studies have been conducted to understand routes of transmission (for a review see references 1 and 29). Lymphoid tissues such as the spleen have been shown to play a fundamental role in agent replication and propagation in the very early stages of disease. Indeed, studies of splenectomized and asplenic mice have shown the lymphoreticular system (LRS) to be an important site for TSE agent replication (14, 26). The periphery also appears to have a role in processing the infectious agent following intracerebral (i.c.) inoculation as PrP^Sc accumulates in the spleen shortly after inoculation and before accumulation of the abnormal protein in the brain (15, 17). Within the LRS, follicular dendritic cells (FDC) have been shown to be important for the uptake of infectivity and subsequent spreading toward the CNS (7, 28, 33, 35). Several studies have also suggested the peripheral nervous systems (PNS) as a potential route of infectivity to the brain, implicating the vagus and sciatic nerves in this process (5, 20, 25, 34).Expression of PrP^C in the peripheral tissues appears to be an important prerequisite for the transport of infectivity to the CNS following peripheral routes of inoculation. Indeed, it has been proposed that a continuous chain of cells expressing PrP^C is fundamental for TSE neuroinvasion (6, 40), with overexpression of endogenous PrP in the PNS greatly facilitating the spread of infectivity (19). Thus, host PrP appears to have a fundamental role in the uptake, transport, and replication of the infectious agent (6). Moreover, it has been suggested that the different PrP^C glycoforms may influence the timing of neuroinvasion by directly influencing the interaction with the infectious agent (19). However, the mechanism by which the different glycoforms are involved in these processes remains to be determined.In order to investigate the role of PrP^C glycosylation in TSE disease after peripheral infection with different TSE strains, we have used our inbred gene-targeted transgenic mice expressing different glycosylated forms of PrP. These mice expressed PrP with no sugars at the first (designated G1/G1 in homozygous mice) or the second glycosylation site (G2/G2) or both (G3/G3) under the control of the endogenous PrP promoter (13). We have previously shown that following intracerebral inoculation, all glycotypes are susceptible to infection with at least one TSE strain and that the type of PrP glycosylation in the host influenced the incubation period but not the distribution of pathological lesions in the brain (45). Here, we examine the influence of host PrP glycosylation on the peripheral acquisition of infection and demonstrate that, unlike the intracerebral route, mice without PrP glycosylation were resistant to disease and that the different glycoforms had a profound influence on not only the timing of disease but also the type and distribution of the PrP^Sc deposits in the brain.     

Effects of including an N-terminal insertion region and arginine-mimetic side chains in helical peptoid analogues of lung surfactant protein B

Surfactant protein B (SP-B) is one of two helical, amphipathic proteins critical for the biophysical functioning of lung surfactant (LS) and hence is an important therapeutic protein. This small, complex 79mer has three internal disulfide bonds and homodimerizes via another disulfide bridge. A helical, amphipathic 25mer from the amino terminus (SP-B(1-25)) exhibits surface-active properties similar to those of full-length, synthetic SP-B. In previous work, we created helical, non-natural mimics of SP-B(1-25) based on sequence-specific peptoid 17mers and demonstrated their biomimetic surface activity. Like SP-B(1-25), the peptoids were designed to adopt helical structures with cationic and nonpolar faces. Here, we compare the surface activities of six different helical peptoid analogues of SP-B(1-25) to investigate the importance of mimicking its N-terminal insertion domain as well as its two arginine residues, both thought to be important for the peptide's proper function. Although the peptoid analogues of SP-B(1-25) studied here share many similar features and all functionally mimic SP-B(1-25) to some degree, it is notable that small differences in their sequences and side chain chemistries lead to substantial differences in their observed interactions with a lipid film. A peptoid comprising a hydrophobic, helical insertion region with aromatic side chains shows more biomimetic surface activity than simpler peptoids, and even better activity, by comparison to natural LS, than SP-B(1-25). However, the substitution of lysine-like side chains for arginine-like side chains in the peptoid has little effect on biomimetic surface activity, indicating that interactions of the guanidino groups with lipids may not be critical for the function of these SP-B mimics.     

Quantitation of Leishmania lipophosphoglycan repeat units by capillary electrophoresis

The glycosylphosphatidylinositol (GPI)-anchored lipophosphoglycan (LPG) of Leishmania is the dominant cell surface glycoconjugate of these pathogenic parasites. LPG is structurally characterized by a series of phosphoglycan repeat units. Determining the number of repeat units per LPG molecule has proven difficult using current technologies, such as mass spectrometry. As an alternative method to quantitate the number of repeat units in LPG, a procedure based on capillary electrophoretic analysis of the proportion of mannose to 2,5-anhydromannose (derived from the nonacetylated glucosamine of the GPI anchor of LPG) was developed. The CE-based technique is sensitive and relatively rapid compared to GC-MS-based protocols. Its application was demonstrated in quantitating the number of LPG repeat units from several species of Leishmania as well as from two life-cycle stages of these organisms.     

Application of Perturbation Simulations in Population Risk Assessment for Different Life History Strategies and Elasticity Patterns

Population structure and life history strategies are determinants of how populations respond to stressor-induced impairments in organism-level responses. Effects on population growth rate were modeled using seven theoretical constructs that represented the life history strategies and elasticity patterns of a broad range of species. Simulations of low to high ranges of simultaneous reductions in survival and reproduction were conducted and results indicated that stressor impacts on population growth rate were a function of population characteristics and the magnitude of the stress. Species that had high reproductive elasticity had greater population-level impacts than species with high survival elasticity for the same organism-level effects. Perturbation simulations were performed to assess the extinction risk in two species with similar elasticity patterns but different life history strategies: mysid shrimp, Americamysis bahia, and the gypsy moth, Lymantria dispar, which can be related to classical K- and r-strategists, respectively. Deterministic extinction risk was greater for the K-strategist, which indicated that population level risks were dependent on life history strategies, and toxicity values (e.g., LC50s) should be interpreted with caution. Ecological risk assessments should consider both population structure and life history strategy of an ecological receptor, in addition to the intrinsic sensitivity of the species to contaminant stressors.     

Elucidation of (-)-epicatechin metabolites after ingestion of chocolate by healthy humans

After absorption in the gastrointestinal tract, (-)-epicatechin is extensively transformed into various conjugated metabolites. These metabolites, chemically different from the aglycone forms found in foods, are the compounds that reach the circulatory system and the target organs. Therefore, it is imperative to identify and quantify these circulating metabolites to investigate their roles in the biological effects associated with (-)-epicatechin intake. Using authentic synthetic standards of (-)-epicatechin sulfates, glucuronides, and O-methyl sulfates, a novel LC-MS/MS-MRM analytical methodology to quantify (-)-epicatechin metabolites in biological matrices was developed and validated. The optimized method was subsequently applied to the analysis of plasma and urine metabolites after consumption of dark chocolate, an (-)-epicatechin-rich food, by humans. (-)-Epicatechin-3'-β-d-glucuronide (C(max) 290±49nM), (-)-epicatechin 3'-sulfate (C(max) 233±60nM), and 3'-O-methyl epicatechin sulfates substituted in the 4', 5, and 7 positions were the most relevant (-)-epicatechin metabolites in plasma. When plasmatic metabolites were divided into their substituent groups, it was revealed that (-)-epicatechin glucuronides, sulfates, and O-methyl sulfates represented 33±4, 28±5, and 33±4% of total metabolites (AUC(0-24)(h)), respectively, after dark chocolate consumption. Similar metabolites were found in urine samples collected over 24h. The total urine excretion of (-)-epicatechin was 20±2% of the amount ingested. In conclusion, we describe the entire metabolite profile and its degree of elimination after administration of (-)-epicatechin-containing food. These results will help us understand more precisely the mechanisms and the main metabolites involved in the beneficial physiological effects of flavanols.