Monoclonal Antibody RYSK173 Recognizes the Dinuclear Zn Center of Serum Carnosinase 1 (CN-1): Possible Consequences of Zn Binding for CN-1 Recognition by RYSK173

Background and Aims

The proportion of serum carnosinase (CN-1) recognized by RYSK173 monoclonal antibody negatively correlates with CN-1 activity. We thus hypothesized that the epitope recognized by RYSK173 is accessible only in a catalytically incompetent conformation of the zinc dependent enzyme and we mapped its position in the CN-1 structure. Since patients with kidney failure are often deficient in zinc and other trace elements we also assessed the RYSK173 CN-1 proportion in serum of these patients and studied the influence of hemodialysis hereon in relation to Zn²⁺ and Cu²⁺ concentration during hemodialysis.

Methods and Results

Epitope mapping using myc-tagged CN-1 fragments and overlapping peptides revealed that the RYSK173 epitope directly contributes to the formation of the dinuclear Zn center in the catalytic domain of homodimeric CN-1. Binding of RYSK173 to CN-1 was however not influenced by addition of Zn²⁺ or Cu²⁺ to serum. In serum of healthy controls the proportion of CN-1 recognized by RYSK173 was significantly lower compared to end-stage renal disease (ESRD) patients (1.12 ± 0.17 vs. 1.56 ± 0.40% of total CN-1; p<0.001). During hemodialysis the relative proportion of RYSK173 CN-1 decreased in parallel with increased serum Zn²⁺ and Cu²⁺ concentrations after dialysis.

Conclusions

Our study clearly indicates that RYSK173 recognizes a sequence within the transition metal binding site of CN-1, thus supporting our hypothesis that metal binding to CN-1 masks the epitope. The CN-1 RYSK173 proportion appears overall increased in ESRD patients, yet it decreases during hemodialysis possibly as a consequence of a relative increase in transition metal bound enzyme.     

Involvement of a Novel Class C Beta-Lactamase in the Transglutaminase Mediated Cross-Linking Cascade of Streptomyces mobaraensis DSM 40847

Streptomyces mobaraensis DSM 40847 secretes transglutaminase that cross-links proteins via γ-glutamyl-ε-lysine isopeptide bonds. Characterized substrates are inhibitory proteins acting against various serine, cysteine and metalloproteases. In the present study, the bacterial secretome was examined to uncover additional transglutaminase substrates. Fractional ethanol precipitation of the exported proteins at various times of culture growth, electrophoresis of the precipitated proteins, and sequencing of a 39 kDa protein by mass spectrometry revealed the novel beta-lactamase Sml-1. As indicated by biotinylated probes, Sml-1, produced in E. coli, exhibits glutamine and lysine residues accessible for transglutaminase. The chromogenic cephalosporin analogue, nitrocefin, was hydrolyzed by Sml-1 with low velocity. The obtained K_m and k_cat values of the recombinant enzyme were 94.3±1.8 μM and 0.39±0.03 s^-1, respectively. Penicillin G and ampicillin proved to be weak inhibitors of nitrocefin hydrolysis (K_i of 0.1 mM and 0.18 mM). Negligible influence of metals on β-lactamase activity ruled out that Sml-1 is a Zn²⁺-dependent class B beta-lactamase. Rather, sequence motifs such as SITK, YSN, and HDG forming the active core in a hypothetical structure may be typical for class C beta-lactamases. Based on the results, we assume that the novel transglutaminase substrate ensures undisturbed growth of aerial hyphae in Streptomyces mobaraensis by trapping and inactivating hostile beta-lactam antibiotics.     

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.     

Intermuscular Coherence in Normal Adults: Variability and Changes with Age

We investigated beta-band intermuscular coherence (IMC) in 92 healthy adults stratified by decade of age, and analysed variability between and within subjects. In the dominant upper limb, IMC was estimated between extensor digitorum communis and first dorsal interosseous as well as between flexor digitorum superficialis and first dorsal interosseous. In the ipsilateral lower limb, IMC was measured between medial gastrocnemius and extensor digitorum brevis as well as between tibialis anterior and extensor digitorum brevis. Age-related changes in IMC were analysed with age as a continuous variable or binned by decade. Intrasession variance of IMC was examined by dividing sessions into pairs of epochs and comparing coherence estimates between these pairs. Eight volunteers returned for a further session after one year, allowing us to compare intrasession and intersession variance. We found no age-related changes in IMC amplitude across almost six decades of age, allowing us to collate data from all ages into an aggregate normative dataset. Interindividual variability ranged over two orders of magnitude. Intrasession variance was significantly greater than expected from statistical variability alone, and intersession variance was even larger. Potential contributors include fluctuations in task performance, differences in electrode montage and short-term random variation in central coupling. These factors require further exploration and, where possible, minimisation. This study provides evidence that coherence is remarkably robust to senescent changes in the nervous system and provides a large normative dataset for future applications of IMC as a biomarker in disease states.     

Alleged Approach-Avoidance Conflict for Food Stimuli in Binge Eating Disorder

ObjectiveFood stimuli are omnipresent and naturally primary reinforcing stimuli. One explanation for the intake of high amounts of food in binge eating disorder (BED) is a deviant valuation process. Valuation of food stimuli is supposed to influence approach or avoidance behaviour towards food. Focusing on self-reported and indirect (facial electromyography) valuation process, motivational aspects in the processing of food stimuli were investigated.MethodsWe compared an overweight sample with BED (BED+) with an overweight sample without BED (BED-) and with normal weight controls (NWC) regarding their self-reported and indirect (via facial electromyography) valuation of food versus non-food stimuli.ResultsRegarding the self-reported valuation, the BED+ sample showed a significantly stronger food-bias compared to the BED- sample, as food stimuli were rated as significantly more positive than the non-food stimuli in the BED+ sample. This self-reported valuation pattern could not be displayed in the indirect valuation. Food stimuli evoked negative indirect valuation in all groups. The BED+ sample showed the plainest approach-avoidance conflict marked by a diverging self-reported (positive) and indirect (negative) valuation of food stimuli.ConclusionsBED+ showed a deviant self-reported valuation of food as compared to BED-. The valuation process of the BED+ sample seems to be characterized by a motivational ambivalence. This ambivalence should be subject of further studies and may be of potential use for therapeutic interventions.     

Gender and the Publication Output of Graduate Students: A Case Study

We examine gender differences among the six PhD student cohorts 2004–2009 at the California Institute of Technology using a new dataset that includes information on trainees and their advisors and enables us to construct detailed measures of teams at the advisor level. We focus on the relationship between graduate student publications and: (1) their gender; (2) the gender of the advisor, (3) the gender pairing between the advisor and the student and (4) the gender composition of the team. We find that female graduate students co-author on average 8.5% fewer papers than men; that students writing with female advisors publish 7.7% more. Of particular note is that gender pairing matters: male students working with female advisors publish 10.0% more than male students working with male advisors; women students working with male advisors publish 8.5% less. There is no difference between the publishing patterns of male students working with male advisors and female students working with female advisors. The results persist and are magnified when we focus on the quality of the published articles, as measured by average Impact Factor, instead of number of articles. We find no evidence that the number of publications relates to the gender composition of the team. Although the gender effects are reasonably modest, past research on processes of positive feedback and cumulative advantage suggest that the difference will grow, not shrink, over the careers of these recent cohorts.     

Strongly Accelerated Margination of Active Particles in Blood Flow

Synthetic nanoparticles and other stiff objects injected into a blood vessel filled with red blood cells are known to marginate toward the vessel walls. By means of hydrodynamic lattice-Boltzmann simulations, we show that active particles can strongly accelerate their margination by moving against the flow direction: particles located initially in the channel center migrate much faster to their final position near the wall than in the nonactive case. We explain our findings by an enhanced rate of collisions between the stiff particles and the deformable red blood cells. Our results imply that a significantly faster margination can be achieved either technically by the application of an external magnetic field (if the particles are magnetic) or biologically by self-propulsion (if the particles are, e.g., swimming bacteria).     

Comparing microscopic counts and pigment analyses in 46 phytoplankton communities from lakes of different trophic state

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