The yeast nucleosome assembly protein 1 (yNap1) plays a role in chromatin maintenance by facilitating histone exchange as well as nucleosome assembly and disassembly. It has been suggested that yNap1 carries out these functions by regulating the concentration of free histones. Therefore, a quantitative understanding of yNap1-histone interactions also provides information on the thermodynamics of chromatin. We have developed quantitative methods to study the affinity of yNap1 for histones. We show that yNap1 binds H2A/H2B and H3/H4 histone complexes with low nM affinity, and that each yNap1 dimer binds two histone fold dimers. The yNap1 tails contribute synergistically to histone binding while the histone tails have a slightly repressive effect on binding. The (H3/H4) 2 tetramer binds DNA with higher affinity than it binds yNap1.Histone chaperones are a diverse group of acidic proteins that bind histones and participate in chromatin assembly and disassembly during replication and transcription. Many members of the various chaperone families play additional and often ill-described roles in cell cycle regulation, apoptosis, and DNA damage repair. Several histone chaperones exhibit tissue-specific functions in transcription regulation (recently reviewed in Refs. 1, 2). As the dynamic nature of nucleosomes and chromatin has become evident, the role of histone chaperones in the modulation of chromatin structure is increasingly recognized. However, little mechanistic insight into the processes of chaperone-mediated nucleosome assembly and disassembly is available. In particular, no quantitative information exists on histone binding, the most fundamental function of histone chaperones.Nucleosome assembly protein 1 (Nap1) 4 was one of the first histone chaperones to be identified (reviewed in Refs. 3, 4). The structure of yeast Nap1 (yNap1) reveals a novel fold that is likely conserved among other members of the Nap1 family (5). At low micromolar concentrations yNap1 exists as a homodimer (6, 7). The large dimer interface is predominantly hydrophobic in character and buries ϳ22% of the overall surface of yNap1 (5); it is therefore unlikely that yNap1 exists in a monomeric state except for perhaps under the most dilute conditions (6). All Nap1 family members have a C-terminal acidic domain (CTAD) of varying length that is not required for histone binding and chromatin assembly, and a variable N-terminal tail of unknown function (3).Nap1 binds all four core histones as well as the linker histone H1 (8 -10). Nap1-mediated nucleosome formation in vitro is characterized by the transfer of a (H3/H4) 2 tetramer onto DNA, followed by the incorporation of H2A/H2B dimer (8). Nakagawa et al. (11) have qualitatively shown that the affinity of the (H3/H4) 2 tetramer for DNA exceeds its affinity for Nap1, and that the affinity of H2A/H2B for a DNA-bound (H3/H4) 2 tetramer (a tetrasome) is greater than its affinity for Nap1.Our understanding of the thermodynamics of chaperonehistone interactions in general, and of Nap1-histone interact...

The organization of skeletal muscles in decapod crustaceans is significantly altered during molting and development. Prior to molting, the claw muscles atrophy dramatically, facilitating their removal from the base of the claw. During development, lobster claw muscles exhibit fiber switching over several molt cycles. Such processes may be influenced by the secretion of steroid molting hormones, known collectively as ecdysteroids. To assay the effects of these hormones, we used eyestalk ablation to trigger an elevation of circulating ecdysteroids and then quantified myofibrillar mRNA levels with real-time PCR and myofibrillar protein levels by SDS-PAGE. Levels of myosin heavy chain (MHC) and actin proteins and the mRNA encoding them were largely unaffected by eyestalk ablation, but in muscles from intact animals, myofibrillar gene expression was modestly elevated in premolt and postmolt animals. In contrast, polyubiquitin mRNA was significantly elevated (about 2-fold) in claw muscles from eyestalk-ablated animals with elevated circulating ecdysteroids. Moreover, patterns of MHC and actin gene expression are significantly different among slow and fast claw muscles. Consistent with these patterns, the three muscle types differed in the relative amounts of myosin heavy chain and actin proteins. All three muscles also co-expressed fast and slow myosin isoforms, even in fibers that are generally regarded as exclusively fast or slow. These results are consistent with other recent data demonstrating co-expression of myosin isoforms in lobster muscles.

Common wheat (Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly (Cephus cinctus, WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography–mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

The objective of the current study was to evaluate the effects of lipid peroxidation products, malondialdehyde (MDA), hexenal, and 4-hydroxynonenal (HNE), on calpain-1 function, and liquid chromatography and tandem mass spectrometry (LC-MS/MS) identification of adducts on calpain-1. Calpain-1 activity slightly increased after incubation with 100 μM MDA but not with 500 and 1000 μM MDA. However, calpain-1 activity was lowered by hexenal and HNE at 100, 500, and 1000 μM. No difference in calpain-1 autolysis was observed between the control and 1000 μM MDA. However, 1000 μM hexenal and HNE treatments slowed the calpain-1 autolysis. Adducts of MDA were detected on glutamine, arginine, lysine, histidine, and asparagine residues via Schiff base formation, while HNE adducts were detected on histidine, lysine, glutamine, and asparagine residues via Michael addition. These results are the first to demonstrate that lipid peroxidation products can impact calpain-1 activity in a concentration-dependent manner and may impact the development of meat tenderness postmortem.

<b><i>Background:</i></b> Assuring adequate antibiotic tissue concentrations at the point of infection, especially in skin and soft tissue infections, is pivotal for an effective treatment and cure. Despite the global issue, a reliable AB monitoring test is missing. Inadequate antibiotic treatment leads to the development of antimicrobial resistances and toxic side effects. β-lactam antibiotics were already detected in sweat of patients treated with the respective antibiotics intravenously before. With the emergence of smartphone-based biosensors to analyse sweat on the spot of need, next-generation molecular digital biomarkers will be increasingly available for a non-invasive pharmacotherapy monitoring. <b><i>Objective:</i></b> Here, we investigated if the glycopeptide antibiotic vancomycin is detectable in sweat samples of in-patients treated with intravenous vancomycin. <b><i>Methods:</i></b> Eccrine sweat samples were collected using the Macroduct Sweat Collector®. Along every sweat sample, a blood sample was taken. Bio-fluid analysis was performed by Ultra-high Pressure Liquid Chromatograph-Tandem Quadrupole Mass Spectrometry coupled with tandem mass spectrometry. <b><i>Results:</i></b> A total of 5 patients were included. Results demonstrate that vancomycin was detected in 5 out of 5 sweat samples. Specifically, vancomycin concentrations ranged from 0.011 to 0.118 mg/L in sweat and from 4.7 to 8.5 mg/L in blood. <b><i>Conclusion:</i></b> Our results serve as proof-of-concept that vancomycin is detectable in eccrine sweat and may serve as a surrogate marker for antibiotic tissue penetration. A targeted vancomycin treatment is crucial in patients with repetitive need for antibiotics and a variable antibiotic distribution such as in peripheral artery disease to optimize treatment effectiveness. If combined with on-skin smartphone-based biosensors and smartphone applications, the detection of antibiotic concentrations in sweat might enable a first digital, on-spot, lab-independent and non-invasive therapeutic drug monitoring in skin and soft tissue infections.