The oligomerization of a family of four genetically clustered human gastrointestinal mucins

Mucins are synthesized and secreted by many epithelia. They are complex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N- glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus- gels.      

Cognitive and functional competence after anaesthesia in patients aged over 60: controlled trial of general and regional anaesthesia for elective hip or knee replacement.

OBJECTIVE--To determine the influence of general or regional anaesthesia on long term mental function in elderly patients. DESIGN--Prospective study of patients randomly allocated to receive general or regional anaesthesia. SETTING--The patients'' homes and a large teaching hospital in Cardiff. SUBJECTS--146 Patients aged 60 and over scheduled for elective hip or knee replacement. MAIN OUTCOME MEASURES--Scores achieved in tests of cognitive function and functional competence. RESULTS--72 Patients were allocated to receive general anaesthesia and 74 regional anaesthesia. Anaesthetic technique did not influence the duration of the operation, time to mobilisation postoperatively, requirements for analgesia after the operation, or duration of stay in hospital. Three months after the operation there was an improvement in the score for the recognition component (76 ms, 95% confidence interval 9 to 144) and the response component (82 ms, 5 to 158) of the choice reaction time in the group receiving general anaesthesia compared with the group receiving regional anaesthesia. This was the only significant difference between the two groups in the assessments of cognitive and functional competence. Eleven patients receiving regional anaesthesia and 12 receiving general anaesthesia reported that their memory and concentration were worse than before the operation, but this was not confirmed by testing. CONCLUSION--Cognitive and functional competence in elderly patients was not detectably impaired after either general or regional anaesthesia when attention was paid to the known perioperative influences on mental function.     

Patch size effects are more important than genetic diversity for plant–herbivore interactions in Brassica crops

1. Considerable evidence suggests that the diversity within plant communities may strongly affect the strength of species interactions, but the majority of studies only considered interspecific diversity. 2. This paper examines the effect of intraspecific genetic diversity within Brassica fields on two Brassica specialists, cabbage root fly, and diamondback moth, and on a parasitoid attacking diamondback moths. Genetic diversity was manipulated both in a replacement and an additive design. 3. Both herbivore densities and parasitism rates were higher in smaller plots, with limited responses to increased within‐plot diversity. All species showed variable densities across genotypes, and preference hierarchies were species specific. 4. Responses to plot size in root flies scaled with the diameter‐to‐area ratio, suggesting that patch detectability affected local density, whereas responses by diamondback moths and parasitoids deviated from this ratio. These species differences could be traced to differences in the residence time within patches, where diamondback moths typically spend longer and more variable time periods in patches than root flies. 5. The lack of response to genetic diversity by both herbivores suggests that egg‐laying rates are affected by decisions on the plant and not by attraction from a distance, neither to the plant itself nor the patch. Patterns of differential attack may then be due to different acceptability for studied genotypes. 6. Future theories on insect responses to spatial heterogeneity should focus on species traits and how traits interact with information landscapes in the field.     

Disturbance mediates the effects of nutrients on developing assemblages of epibiota

Local dynamics such as resource enhancement (e.g. nutrient supply) and stochastic events of destruction (disturbances that provide new space) are hypothesized to counteractively affect species diversity and composition. We tested the independent and interactive effects of nutrients and disturbance on the development of assemblages of epibiota attached to vertical surfaces in an oligotrophic system. Nutrient concentrations were manipulated at three levels (ambient, medium and high) while disturbance was manipulated by removing biomass at seven frequencies (0×, 2×, 3×, 4×, 5×, 7×, 12×). Nutrient and disturbance regimes had opposing effects on diversity such that species richness increased with resource enhancement (nutrients) and declined with disturbance. These results support the model that increased heterogeneity of distribution of limiting resources allows the coexistence of species with low and high resource requirements.     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Biogeography and population structure of the Neotropical endemic yeast species <Emphasis Type="Italic">Metschnikowia lochheadii</Emphasis>

The genetic structure of populations of the out-crossing haplontic yeast species Metschnikowia lochheadii was investigated. The species is associated with floricolous beetles in Central America and Hawaii. The objective was to determine whether sexual reproduction is prevalent and to what extent the geographic distribution of genotypes can be viewed as historical. The genetic markers examined include the mating type (h (+) or h (-)) and nine polymorphic DNA loci. The data were used to assess population structuring based on F (ST) and linkage disequilibrium and the distribution of alleles using parsimony haplotype networks. In Central America, M. lochheadii is subdivided into sexually active demes between which gene flow is limited. Isolates from five Hawaiian islands had identical haplotypes, confirming that the species has undergone a founder effect concomitant with the recent import of a nitidulid beetle into the archipelago.     

Analysis of 'driver' and 'passenger' CD8+ T-cell responses against variable viruses

Variable viruses, such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV), persist despite host immune responses directed against them. Numerous lines of evidence have suggested that antiviral CD8+ T-cell responses are key among these immune responses, but these vary widely in their ability to contain virus. We propose that only a proportion of responses may exert significant antiviral pressure ('driver' responses), leading to control over viral replication (protection) and/or, ultimately, selection of escape mutants. Another set of responses may exert only weak pressure on the virus ('passenger' responses): these neither protect nor select. To examine this we have analysed (using established databases of HIV and HCV sequences and cytotoxic T-lymphocyte (CTL) epitopes, and published experimental datasets) two important features--predicted binding of the epitope to major histocompatibility complex molecule and observed variability of the epitope--that might distinguish such responses. We find that a high predicted binding estimate could only explain a limited set of 'driver' responses associated with protection or selection. There is statistical evidence that readily defined (and non-protective) CTL responses target regions associated with lower levels of viral variability. Taken together, this suggests that a large number of well-documented responses may represent 'passengers' and we propose a mechanism that might explain their presence.     

Impacts of multiple anthropogenic stressors on stream macroinvertebrate community composition and functional diversity

Ensuring the provision of essential ecosystem services in systems affected by multiple stressors is a key challenge for theoretical and applied ecology. Trait‐based approaches have increasingly been used in multiple‐stressor research in freshwaters because they potentially provide a powerful method to explore the mechanisms underlying changes in populations and communities. Individual benthic macroinvertebrate traits associated with mobility, life history, morphology, and feeding habits are often used to determine how environmental drivers structure stream communities. However, to date multiple‐stressor research on stream invertebrates has focused more on taxonomic than on functional metrics. We conducted a fully crossed, 4‐factor experiment in 64 stream mesocosms fed by a pristine montane stream (21 days of colonization, 21 days of manipulations) and investigated the effects of nutrient enrichment, flow velocity reduction and sedimentation on invertebrate community, taxon, functional diversity and trait variables after 2 and 3 weeks of stressor exposure. 89% of the community structure metrics, 59% of the common taxa, 50% of functional diversity metrics, and 79% of functional traits responded to at least one stressor each. Deposited fine sediment and flow velocity reduction had the strongest impacts, affecting invertebrate abundances and diversity, and their effects translated into a reduction of functional redundancy. Stressor effects often varied between sampling occasions, further complicating the prediction of multiple‐stressor effects on communities. Overall, our study suggests that future research combining community, trait, and functional diversity assessments can improve our understanding of multiple‐stressor effects and their interactions in running waters.     

Requirements for Hirano Body Formation

Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point mutation within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the microtubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano bodies.