Opposite Contributions of Trunk and Leg Fat Mass with Plasma Lipase Activities: The Hoorn Study

Objective: Lipoprotein lipase (LPL) and hepatic lipase (HL) are essential in hydrolysis of triglyceride‐rich lipoproteins. LPL activity is negatively, whereas HL activity is positively, associated with total body fat. We determined the associations of trunk and leg fat mass with plasma LPL and HL activities in a cross‐sectional study. Research Methods and Procedures: LPL and HL activities were determined in post‐heparin plasma in a sample of 197 men and 209 women, 60 to 87 years of age. A total body DXA scan was performed to determine trunk and leg fat mass. Results: In women, but not in men, trunk fat mass was negatively associated with LPL activity, whereas leg fat mass was positively associated, after mutual adjustment and adjustment for age. Standardized βs (95% confidence interval) for trunk and leg fat mass were ?0.24 (?0.41; ?0.08) and 0.14 (?0.02; 0.31), respectively (interaction by sex, p = 0.03). Larger trunk fat mass was associated with higher HL activity in men [0.48 (0.28; 0.68)] and women [0.40 (0.24; 0.56)]. A negative association of leg fat mass and HL activity was observed in men, although not statistically significant [?0.13 (?0.33; 0.06)], and in women [?0.28 (?0.38; ?0.18)]. Discussion: Abdominal fat is associated with unfavorable and femoral fat with favorable LPL and HL activities in plasma.     

Human Anti-Varicella-Zoster Virus (VZV) Recombinant Monoclonal Antibody Produced after Zostavax Immunization Recognizes the gH/gL Complex and Neutralizes VZV Infection

Varicella-zoster virus (VZV) is a ubiquitous, highly cell-associated, and exclusively human neurotropic alphaherpesvirus. VZV infection is initiated by membrane fusion, an event dependent in part on VZV glycoproteins gH and gL. Consistent with its location on the virus envelope, the gH/gL complex is a target of neutralizing antibodies produced after virus infection. One week after immunizing a 59-year-old VZV-seropositive man with Zostavax, we sorted his circulating blood plasma blasts and amplified expressed immunoglobulin variable domain sequences by single-cell PCR. Sequence analysis identified two plasma blast clones, one of which was used to construct a recombinant monoclonal antibody (rec-RC IgG). The rec-RC IgG colocalized with VZV gE on the membranes of VZV-infected cells and neutralized VZV infection in tissue culture. Mass spectrometric analysis of proteins immunoprecipitated by rec-RC IgG identified both VZV gH and gL. Transfection experiments showed that rec-RC IgG recognized a VZV gH/gL protein complex but not individual gH or gL proteins. Overall, our recombinant monoclonal anti-VZV antibody effectively neutralizes VZV and recognizes a conformational epitope within the VZV gH/L protein complex. An unlimited supply of this antibody provides the opportunity to analyze membrane fusion events that follow virus attachment and to identify multiple epitopes on VZV-specific proteins.     

Flow cytometric analysis of Lactobacillus plantarum to monitor lag times, cell divisionand injury

Flow cytometry in combination with fluorescent molecular markers 5- (and 6-)carboxyfluorescein succinimidylester (CFSE) and propidium iodide (PI) have been applied todetermine lag times, numbers of cell divisions and injury after mild heat (50°C, 5 min) andnisin treatments (0·1 and 1·0 μg ml⁻¹) of Lactobacillus plantarum. Initial labelling with covalently bound dye CFSE (20 and 100 μg ml⁻¹)allowed determination of lag times and cell proliferation for up to eight generations.Double-labelling with CFSE and PI (5 μg ml⁻¹) provided additional informationabout damage levels and distributions within populations. Subpopulations surviving treatmentcould be identified easily and selectively sorted.     

Strategies to assess and optimize stability of endogenous amines during cerebrospinal fluid sampling

Introduction

Metabolic profiling of cerebrospinal fluid (CSF) is a promising technique for studying brain diseases. Measurements should reflect the in vivo situation, so ex vivo metabolism should be avoided.

Objective

To investigate the effects of temperature (room temperature vs. 4 °C), centrifugation and ethanol, as anti-enzymatic additive during CSF sampling on concentrations of glutamic acid, glutamine and other endogenous amines.

Methods

CSF samples from 21 individuals were processed using five different protocols. Isotopically-labeled alanine, isoleucine, glutamine, glutamic acid and dopamine were added prior to sampling to trace any degradation. Metabolomics analysis of endogenous amines, isotopically-labeled compounds and degradation products was performed with a validated LC–MS method.

Results

Thirty-six endogenous amines were quantified. There were no statistically significant differences between sampling protocols for 31 out of 36 amines. For GABA there was primarily an effect of temperature (higher concentrations at room temperature than at 4 °C) and a small effect of ethanol (lower concentrations if added) due to possible degradation. O-phosphoethanolamine concentrations were also lower when ethanol was added. Degradation of isotopically-labeled compounds (e.g. glutamine to glutamic acid) was minor with no differences between protocols.

Conclusion

Most amines can be considered stable during sampling, provided that samples are cooled immediately to 4 °C, centrifuged, and stored at ??80 °C within 2 h. The effect of ethanol addition for more unstable metabolites needs further investigation. This was the first time that labeled compounds were used to monitor ex vivo metabolism during sampling. This is a useful strategy to study the stability of other metabolites of interest.

     

Evidence for diet partitioning among three coexisting native freshwater fishes in South Africa's Cape Fold Ecoregion

The partitioning of limited resources commonly explains how different species can coexist within the same ecological community. In this 2010 study, the diets of three coexisting freshwater fishes (Cape galaxias Galaxias zebratus, n = 27; Cape kurper Sandelia capensis, n = 60; Breede River redfin Pseudobarbus burchelli, n = 77) were characterised and compared in three headwater streams in South Africa's Cape Fold Ecoregion using gut contents and stable isotope analyses. These data were analysed to ascertain whether the three species exploit distinct trophic niches. Both approaches provided evidence that these species occupy different trophic niches, though with some overlap. However, dietary differences among sites were not consistent and were probably influenced by site-specific factors like resource availability. Pseudobarbus burchelli had a broader niche breadth at Tierkloof Stream than the other two species, but not at Waaihoek or Tierstel Streams. Our results also suggest that P. burchelli consumed a more omnivorous diet than do the other two species, whereas S. capensis occupied a higher trophic position than the other two species and consumed vertebrates. Our findings suggest that these species occupy non-equivalent feeding niches in Cape Fold Ecoregion headwater streams, and that diet partitioning might facilitate their coexistence in these systems.     

Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

Identification of wear particles of joint prostheses in tissues using laser microprobe mass analysis (LAMMA)]

The definite identification of wear particles from joint prostheses is of great importance for the development of joint replacement, as the type and quantity of different wear particles gives information on the wear resistance of implant materials. From the types of prostheses nowadays in use polyethylene wear of the sockets, bone cement wear, metallic and ceramic wear can be generated. Whereas polyethylene wear can be easily identified by its bright luminescence in polarized light and its characteristic configuration, the distinction of the small granular wear particles of the bone cement, metal and ceramic by light microscope is difficult. The laser microprobe mass analysis (LAMMA) is a method, which allows the analysis of single light microscopically detectable wear particles in tissues. Not only contrast medium particles of the bone cements (zirconium oxide or barium sulfate) but also metallic and aluminum oxide particles could be definitely identified within the pseudocapsules as well as in regional lymph nodes by LAMMA-analysis, whereby the bone cement wear predominated. In addition, the distinction between organic substances (as blood degradation products), which may appear similar to wear particles in configuration and colour, and the foreign material is also possible with this method.