Galactosyl transferase of a Golgi fraction from cultured neoplastic mast cells

Suspension cultures of neoplastic mouse mast cells were used to obtain large quantities of a homogeneous cell population as starting material for cell fractionation. A Golgi fraction was prepared by slight modification of established techniques and identified by electron microscopy. Assay of galactosyl transferase activity using ovalbumin, desialylated degalactosylated orosomucoid, and N-acetylglucosamine as galactose acceptors showed that the Golgi fraction was enriched in specific activity over the homogenate. The Golgi galactosyl transferase was examined in detail. Acceptor concentrations for optimal galactose incorporation were determined, and substrate inhibition effects were shown with higher concentrations of all three acceptors. Manganese was shown to be necessary for galactose incorporation. A higher concentration of manganese afforded some protection from substrate inhibition by acceptors, but at the same time was itself inhibitory. All three acceptors competed with one another for galactose incorporation, indicating that a single enzyme catalyzed the transfer of galactose for all acceptors.     

A micro method for simultaneous determination of galactosyltransferase and 5''-nucleotidase activities in cell fractions.

Galactosyltransferase and 5'-nucleotidase were assayed in the same reaction mixture, with ovalbumin as exogenous acceptor of (14-C)galactose and with (3-H)AMP as the substrate for the 5'-nucleotidase assay. The substrates and reaction products of either assay had no significant effect on the activity of the other enzyme.     

Spatio‐temporal scaling of biodiversity and the species–time relationship in a stream fish assemblage

1. The increase of species richness with the area of the habitat sampled, that is the species–area relationship, and its temporal analogue, the species–time relationship (STR), are among the few general laws in ecology with strong conservation implications. However, these two scale‐dependent phenomena have rarely been considered together in biodiversity assessment, especially in freshwater systems. 2. We examined how the spatial scale of sampling influences STRs for a Central‐European stream fish assemblage (second‐order Bernecei stream, Hungary) using field survey data in two simulation‐based experiments. 3. In experiment one, we examined how increasing the number of channel units, such as riffles and pools (13 altogether), and the number of field surveys involved in the analyses (12 sampling occasions during 3 years), influence species richness. Complete nested curves were constructed to quantify how many species one observes in the community on average for a given number of sampling occasions at a given spatial scale. 4. In experiment two, we examined STRs for the Bernecei fish assemblage from a landscape perspective. Here, we evaluated a 10‐year reach level data set (2000–09) for the Bernecei stream and its recipient watercourse (third‐order Kemence stream) to complement results on experiment one and to explore the mechanisms behind the observed patterns in more detail. 5. Experiment one indicated the strong influence of the spatial scale of sampling on the accumulation of species richness, although time clearly had an additional effect. The simulation methodology advocated here helped to estimate the number of species in a diverse combination of spatial and temporal scale and, therefore, to determine how different scale combinations influence sampling sufficiency. 6. Experiment two revealed differences in STRs between the upstream (Bernecei) and downstream (Kemence) sites, with steeper curves for the downstream site. Equations of STR curves were within the range observed in other studies, predominantly from terrestrial systems. Assemblage composition data suggested that extinction–colonisation dynamics of rare, non‐resident (i.e. satellite) species influenced patterns in STRs. 7. Our results highlight that the determination of species richness can benefit from the joint consideration of spatial and temporal scales in biodiversity inventory surveys. Additionally, we reveal how our randomisation‐based methodology may help to quantify the scale dependency of diversity components (α, β, γ) in both space and time, which have critical importance in the applied context.     

NetCooperate: a network-based tool for inferring host-microbe and microbe-microbe cooperation

Background

Host-microbe and microbe-microbe interactions are often governed by the complex exchange of metabolites. Such interactions play a key role in determining the way pathogenic and commensal species impact their host and in the assembly of complex microbial communities. Recently, several studies have demonstrated how such interactions are reflected in the organization of the metabolic networks of the interacting species, and introduced various graph theory-based methods to predict host-microbe and microbe-microbe interactions directly from network topology. Using these methods, such studies have revealed evolutionary and ecological processes that shape species interactions and community assembly, highlighting the potential of this reverse-ecology research paradigm.

Results

NetCooperate is a web-based tool and a software package for determining host-microbe and microbe-microbe cooperative potential. It specifically calculates two previously developed and validated metrics for species interaction: the Biosynthetic Support Score which quantifies the ability of a host species to supply the nutritional requirements of a parasitic or a commensal species, and the Metabolic Complementarity Index which quantifies the complementarity of a pair of microbial organisms’ niches. NetCooperate takes as input a pair of metabolic networks, and returns the pairwise metrics as well as a list of potential syntrophic metabolic compounds.

Conclusions

The Biosynthetic Support Score and Metabolic Complementarity Index provide insight into host-microbe and microbe-microbe metabolic interactions. NetCooperate determines these interaction indices from metabolic network topology, and can be used for small- or large-scale analyses. NetCooperate is provided as both a web-based tool and an open-source Python module; both are freely available online at http://elbo.gs.washington.edu/software_netcooperate.html.     

The effect of rFVIIa on pro- and anti-inflammatory cytokines in serum and cerebrospinal fluid in a swine model of traumatic brain injury

Traumatic brain injury (TBI) is associated with significant infectious and inflammatory complications. Though increasing evidence suggests that rFVIIa administration may be efficacious for the pre-hospital treatment of TBI, the FVIIa-tissue factor complex has been shown to be immunologically active. To date the cytokine response to rFVIIa administration for the treatment of TBI has not been evaluated. Twenty anesthetized immature Yorkshire swine underwent fluid percussion TBI. At 15 min following injury, animals were randomized to receive either 90 μg/kg rFVIIa (rFVIIa) or nothing. Animals were observed for 6 h and then euthanized. Plasma and cerebrospinal (CSF) samples were collected at 0 min and 360 min, and ELISA analysis of TNF-α, IL-1β and IL-10 was performed. Survival in both groups was 100%. Baseline cytokine concentrations were not statistically different between rFVIIa and control animals in plasma or CSF. Animals in both groups did not have significant changes in plasma cytokine concentrations following TBI. Control animals did not demonstrate significant changes from baseline of CSF cytokine concentrations following TBI. The administration of rFVIIa however, resulted in significant increases in CSF TNF-α concentration (232.0 pg/ml ± 75.9 vs 36.4 pg/ml ± 10.4, p = 0.036) and IL-10 concentration (10.7 pg/ml ± 0.6 vs 8.8 pg/ml ± 0.1, p = 0.015). IL-1β concentrations were not significantly changed over the experimental time course. These results suggest that rFVIIa administration for the treatment of TBI is not immunologically inert, and is associated with increased CSF concentrations of TNF-α and IL-10.     

Regional muscle oxygenation differences in vastus lateralis during different modes of incremental exercise

Background

Near infrared spectroscopy (NIRS) is used to assess muscle oxygenation (MO) within skeletal muscle at rest and during aerobic exercise. Previous investigations have used a single probe placement to measure MO during various forms of exercise. However, regional MO differences have been shown to exist within the same muscle which suggests that different areas of the same muscle may have divergent MO. Thus, the aim of this study was to examine whether regional differences in MO exist within the same muscle during different types of incremental (rest, 25, 50, 75, 100 % of maximum) exercise (1 leg knee extension (KE), 2 leg KE, or cycling).

Methods

Nineteen healthy active males (Mean ± SD: Age 27 ± 4 yrs; VO_2max: 55 ± 11 mL/kg/min) performed incremental exercise to fatigue using each mode of exercise. NIRS probes were placed on the distal and proximal portion of right leg vastus lateralis (VL). Results were analyzed with a 3-way mixed model ANOVA (probe × intensity × mode).

Results

Differences in MO exist within the VL for each mode of exercise, however these differences were not consistent for each level of intensity. Comparison of MO revealed that the distal region of VL was significantly lower throughout KE exercise (1 leg KE proximal MO – distal MO = 9.9 %; 2 leg KE proximal MO – distal MO = 13 %). In contrast, the difference in MO between proximal and distal regions of VL was smaller in cycling and was not significantly different at heavy workloads (75 and 100 % of maximum).

Conclusion

MO is different within the same muscle and the pattern of the difference will change depending on the mode and intensity of exercise. Future investigations should limit conclusions on MO to the area under assessment as well as the type and intensity of exercise employed.

     

Association of FCGR2A and FCGR2A-FCGR3A haplotypes with susceptibility to giant cell arteritis

The Fc gamma receptors have been shown to play important roles in the initiation and regulation of many immunological and inflammatory processes and to amplify and refine the immune response to an infection. We have investigated the hypothesis that polymorphism within the FCGR genetic locus is associated with giant cell arteritis (GCA). Biallelic polymorphisms in FCGR2A, FCGR3A, FCGR3B and FCGR2B were examined for association with biopsy-proven GCA (n = 85) and healthy ethnically matched controls (n = 132) in a well-characterised cohort from Lugo, Spain. Haplotype frequencies and linkage disequilibrium (D') were estimated across the FCGR locus and a model-free analysis performed to determine association with GCA. There was a significant association between FCGR2A-131RR homozygosity (odds ratio (OR) 2.10, 95% confidence interval (CI) 1.12 to 3.77, P = 0.02, compared with all others) and carriage of FCGR3A-158F (OR 3.09, 95% CI 1.10 to 8.64, P = 0.03, compared with non-carriers) with susceptibility to GCA. FCGR haplotypes were examined to refine the extent of the association. The haplotype showing the strongest association with GCA susceptibility was the FCGR2A-FCGR3A 131R-158F haplotype (OR 2.84, P = 0.01 for homozygotes compared with all others). There was evidence of a multiplicative joint effect between homozygosity for FCGR2A-131R and HLA-DRB1*04 positivity, consistent with both of these two genetic factors contributing to the risk of disease. The risk of GCA in HLA-DRB1*04 positive individuals homozygous for the FCGR2A-131R allele is increased almost six-fold compared with those with other FCGR2A genotypes who are HLA-DRB1*04 negative. We have demonstrated that FCGR2A may contribute to the 'susceptibility' of GCA in this Spanish population. The increased association observed with a FCGR2A-FCGR3A haplotype suggests the presence of additional genetic polymorphisms in linkage disequilibrium with this haplotype that may contribute to disease susceptibility. These findings may ultimately provide new insights into disease pathogenesis.