Inhibition of human muscle-specific enolase by methylglyoxal and irreversible formation of advanced glycation end products

Methylglyoxal (MG) was studied as an inhibitor and effective glycating factor of human muscle-specific enolase. The inhibition was carried out by the use of a preincubation procedure in the absence of substrate. Experiments were performed in anionic and cationic buffers and showed that inhibition of enolase by methylglyoxal and formation of enolase-derived glycation products arose more effectively in slight alkaline conditions and in the presence of inorganic phosphate. Incubation of 15 micromolar solutions of the enzyme with 2 mM, 3.1 mM and 4.34 mM MG in 100 mM phosphate buffer pH 7.4 for 3 h caused the loss a 32%, 55% and 82% of initial specific activity, respectively. The effect of MG on catalytic properties of enolase was investigated. The enzyme changed the K_M value for glycolytic substrate 2-phospho-D-glycerate (2-PGA) from 0.2 mM for native enzyme to 0.66 mM in the presence of MG. The affinity of enolase for gluconeogenic substrate phosphoenolpyruvate altered after preincubation with MG in the same manner, but less intensively. MG has no effect on V_max and optimal pH values. Incubation of enolase with MG for 0-48 h generated high molecular weight protein derivatives. Advanced glycation end products (AGEs) were resistant to proteolytic degradation by trypsin. Magnesium ions enhanced the enzyme inactivation by MG and facilitated AGEs formation. However, the protection for this inhibition in the presence of 2-PGA as glycolytic substrate was observed and AGEs were less effectively formed under these conditions.     

The Molecular Basis of Conformational Instability of the Ecdysone Receptor DNA Binding Domain Studied by In Silico and In Vitro Experiments

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule.     

Multistage aqueous two‐phase extraction of a monoclonal antibody from cell supernatant

This article presents results of continuous multistage aqueous two‐phase extraction of an immunoglobulin G1 from cell supernatant in a mixer‐settler unit. An aqueous two‐phase system consisting of polyethylene glycol 2000, phosphate salt, and water was applied without and with sodium chloride (NaCl). Influences of different parameters such as throughput, phase ratio, and stage number on the extraction performance were analyzed. For systems without NaCl, the extraction was carried out as a washing step. An increase of stage number from one to five stages enabled to increase the immunoglobulin G1 purity from 11.8 to 32.6% at a yield of nearly 90%. Furthermore, a reduction of product phase volume due to a higher phase ratio led to an increase of purity from 20.8 to 29.6% in a three‐stage countercurrent extraction. For experiments with NaCl moderate partitioning conditions were adjusted by adding 8 wt% NaCl. In that case, the extraction was carried out as a stripping step. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:925–936, 2015     

The Relationship of Metabolic Syndrome with Stress,Coronary Heart Disease and Pulmonary Function - An Occupational Cohort-Based Study

Background and Aims

Higher levels of stress impact the prevalence of metabolic syndrome (MetS) and coronary heart disease. The association between MetS, impaired pulmonary function and low level of physical activity is still pending assessment in the subjects exposed to stress. The study aimed to examine whether higher levels of stress might be related to MetS and the plaque presence, as well as whether MetS might affect pulmonary function.

Design and Methods

The study embraced 235 police officers (mean age 40.97 years) from the south of Poland. The anthropometrics and biochemical variables were measured; MetS was diagnosed using the International Diabetes Federation criteria. Computed tomography coronary angiography of coronary arteries, exercise ECG, measurements of brachial flow-mediated dilation, and carotid artery intima-media thickness were completed. In order to measure the self-perception of stress, 10-item Perceived Stress Scale (PSS-10) was applied. Pulmonary function and physical activity levels were also addressed. Multivariate logistic regression analyses were applied to determine the relationships between: 1/ incidence of coronary plaque and MetS per se, MetS components and the number of classical cardiovascular risk factors, 2/ perceived stress and MetS, 3/ MetS and pulmonary function parameters.

Results

Coronary artery atherosclerosis was less associated with MetS (OR = 2.62, 95%CI 1.24–5.52; p = 0.011) than with a co-existence of classical cardiovascular risk factors (OR = 5.67, 95% CI 1.07–29.85, p = 0.03; for 3 risk factors and OR = 9.05; 95% CI 1.24–66.23, p = 0.02; for 6 risk factors, respectively). Perceived stress increased MetS prevalence (OR = 1.07, 95% CI 1.03–1.13; p = 0.03), and impacted coronary plaque prevalence (OR = 1.05, 95% CI 1.001–1.10; p = 0.04). Leisure-time physical activity reduced the chances of developing MetS (OR = 0.98 95% CI 0.96–0.99; p = 0.02). MetS subjects had significantly lower values of certain pulmonary function parameters.

Conclusions

Exposure to job-specific stress among police officers increased the prevalence of MetS and impacted coronary plaque presence. MetS subjects had worse pulmonary function parameters. Early-stage, comprehensive therapeutic intervention may reduce overall risk of cardiovascular events and prevent pulmonary function impairment in this specific occupational population.     

Static Postural Stability in Women during and after Pregnancy: A Prospective Longitudinal Study

This longitudinal study aimed to compare static postural stability in women between early pregnancy, advanced pregnancy, and at 2 and 6 months postpartum. Forty-five pregnant women were enrolled and 31 completed the protocol. Data were collected at 7–16 and 34–39 weeks gestation, and at 6–10 and 26–30 weeks postpartum. For each subject, the center of foot pressure path length and mean velocity (with directional subcomponents) were computed from 30-s long quiet-standing trials on a stationary force plate with eyes open or closed. The body mass, stance width, and sleep duration within 24 h before testing were also recorded. Static postural stability was not different between pregnancy and postpartum, except for the anterior posterior sway tested in the eyes-closed condition, which was significantly increased in late pregnancy compared to that at 2 and 6 months postpartum. Pregnant/postpartum women’s body mass weakly positively correlated with anterior-posterior sway in the eyes-closed condition and their stance width weakly positively correlated with the anterior-posterior sway in the eyes-open condition. No effect of sleep duration on postural sway was found. Our findings indicate that under visual deprivation conditions women in advanced pregnancy may have decreased static stability compared to their non-pregnant state.     

Silk as an innovative biomaterial for cancer therapy

Silk has been used for centuries in the textile industry and as surgical sutures. In addition to its unique mechanical properties, silk possesses other properties, such as biocompatibility, biodegradability and ability to self-assemble, which make it an interesting material for biomedical applications. Although silk forms only fibers in nature, synthetic techniques can be used to control the processing of silk into different morphologies, such as scaffolds, films, hydrogels, microcapsules, and micro- and nanospheres. Moreover, the biotechnological production of silk proteins broadens the potential applications of silk. Synthetic silk genes have been designed. Genetic engineering enables modification of silk properties or the construction of a hybrid silk. Bioengineered hybrid silks consist of a silk sequence that self-assembles into the desired morphological structure and the sequence of a polypeptide that confers a function to the silk biomaterial. The functional domains can comprise binding sites for receptors, enzymes, drugs, metals or sugars, among others. Here, we review the current status of potential applications of silk biomaterials in the field of oncology with a focus on the generation of implantable, injectable and targeted drug delivery systems and the three-dimensional cancer models based on silk scaffolds for cancer research. However, the systems described could be applied in many biomedical fields.     

Homodimerization propensity of the intrinsically disordered N-terminal domain of Ultraspiracle from Aedes aegypti

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD–NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.