The Comparison of Physicochemical Parameters,Antioxidant Activity and Proteins for the Raw Local Polish Honeys and Imported Honey Blends

Many imported honeys distributed on the Polish market compete with local products mainly by lower price, which can correspond to lower quality and widespread adulteration. The aim of the study was to compare honey samples (11 imported honey blends and 5 local honeys) based on their antioxidant activity (measured by DPPH, FRAP, and total phenolic content), protein profile obtained by native PAGE, soluble protein content, diastase, and acid phosphatase activities identified by zymography. These indicators were correlated with standard quality parameters (water, HMF, pH, free acidity, and electrical conductivity). It was found that raw local Polish honeys show higher antioxidant and enzymatic activity, as well as being more abundant in soluble protein. With the use of principal component analysis (PCA) and stepwise linear discriminant analysis (LDA) protein content and diastase number were found to be significant (p < 0.05) among all tested parameters to differentiate imported honey from raw local honeys.     

Quantitative predictions for DNA two-dimensional display according to size and nucleotide sequence composition

2-D DNA display is a simple separation method that provides a fast and economical way of visualizing polymorphism and comparing genomes. The DNA fragments are separated first according to their size by standard gel electrophoresis and then according to their sequence composition using denaturing gradient gel electrophoresis. First developed by Fischer and Lerman (Cell 1979, 16, 191-200), this method has recently been used to distinguish strains within a bacterial species. The genomic restriction fragments are displayed as spots on a 2-D surface. Although most of the relevant physical mechanisms are understood, this technique is mostly empirical and remains essentially qualitative. In view of optimizing this procedure, we combine our understanding of the different physical mechanisms at play to develop a complete numerical model to predict the relative coordinates of the spots as a function of the corresponding DNA sequence and of the experimental conditions. We experimentally validate our model by predicting the outcome of a 2-D display of the lambda phage genome. It thus becomes possible to optimize in silico the experimental parameters, to predict whether specific mutations as well as yet undescribed genetic polymorphisms can be resolved, and to assist in interpreting the experimental data.     

The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis

Nucleoside diphosphate kinase (NDPK) has been shown to play a pivotal role in modulating a plethora of cellular processes. In this study, we report on a blue native (BN) PAGE technique which allows the facile assessment of NDPK activity and expression. The in-gel detection of NDPK relies on the precipitation of formazan at the site of immobilized enzyme activity. This is achieved by coupling the formation of ATP, as a consequence of gamma-phosphate transfer from NTP to ADP, to hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), oxidized nicotinamide adenine dinucleotide phosphate (NADP), phenazine methosulfate (PMS), and iodonitrotetrazolium chloride (INT). 2-D denaturing gel analysis confirmed that the activity bands corresponded to NDPK as indicated by subunit composition. Furthermore, the sensitivity and specificity of this readily accessible procedure was assessed by monitoring the in-gel activity of NDPK using different concentrations of GTP and CTP as well as deoxynucleoside triphosphates. This electrophoretic technique allows the quick and easy detection of NDPK, a housekeeping enzyme crucial to cell survival.     

Detection of recombinant growth hormone in human plasma by a 2‐D PAGE method

Human growth hormone (GH) has several central metabolic functions including bone growth in childhood, and its anabolic and lipolytic effects in particular are assumed reasons for the abuse of GH by athletes. Human endogenous GH consists of a main 22 kDa variant and several isoforms. In contrast, recombinant GH consists of only one variant being identical to the main endogenous isoform. The method presented here separates different isoforms by 2‐D PAGE after isolation of GH from plasma using an immunoaffinity purification system. While samples containing endogenous GH yield up to four isoforms, samples with recombinant GH contain the main 22 kDa spot only. Normalized spot volumes (NSV) are calculated after addition of an internal standard and a discrimination limit was determined at 0.52 for the NSV of the main 22 kDa spot. Above this value, samples containing endogenous GH show at least the main 22 kDa isoform and the 20 kDa splice variant. In contrast, samples with a NSV >0.52 and only one spot are suspicious to contain recombinant GH. This method detects discrete isoforms of GH from plasma and discriminates endogenous GH from its recombinant analog, which makes it useful for doping control purposes.     

Urine protein quantification in stacking gel by SDS‐PAGE

Urine total protein concentration is usually measured by the pyrogallol red‐molybdate (PRM) assay in clinical laboratories, but it is often subject to sample interference. Here, we introduce a stacking gel‐based method for accurate protein quantitation. In this method, the urine protein samples are run into the stacking gel by SDS‐PAGE where it is concentrated into a single band, and then quickly stained by 0.001% Coomassie at high temperature. High correlations were found between the BSA and urine protein standards (R² = 0.997 and 0.990, respectively). Addition of 80 ng urine protein standard into each of the ten clinical urine specimens with questionable PRM results yielded the expected increase in the results by this method. Comparison of the PRM method and with the gel quantitation approach on about 60 clinical urine samples demonstrated a general consistency between the results (R² = 0.825), but in PRM samples with lower protein concentration showed more variations. Overall, the stacking gel method might be a good alternative for clinical urine samples with suspicious protein concentration results.     

Comparative Electrochemical and Spectroscopic Studies of I‐Motif‐forming DNA Nonamers

The paper shows the structural diversity of cytosine (C)‐rich oligodeoxynucleotides (ODNs) arising from their detail nucleotide sequence and experimental conditions. In slightly acidic solutions, the ODN nonamers with different adenine (A) and cytosine (C) sequences can adopt non‐canonical structures involving protonated bases. A distinct secondary structure formed in (C)‐rich sequences, called i‐motif (iM), consists of hemiprotonated and intercalated cytosine base pairs (C.C⁺). Folding and unfolding of particular structures in solutions were monitored by ¹H NMR and CD spectroscopies and native polyacrylamide gel electrophoresis (PAGE), which are capable to determine their structural characteristics. Effects of sequences and their proclivity to formation of the iM on electrochemical behaviour of the ODN nonamers were studied by electrochemical methods. The LSV signals of A and C obtained from the reductive dissolution of ODN adsorption layers on a hanging mercury drop electrode were processed by elimination voltammetry with linear scan (EVLS), which revealed complex effects of the nonamer properties (namely their primary and secondary structure confirmed in solution) on their adsorption and reduction activity.     

两种多酸型酪氨酸酶抑制剂的性能研究

以H₃PW₁₂O₄₀和H₄SiW₁₂O₄₀(简写为PW₁₂和SiW₁₂)为效应物, 测定其对酪氨酸酶活力的抑制作用. 通过非变性聚丙烯凝胶(Native-PAGE)电泳确定酪氨酸酶是多家族基因编码, 其分子量为3×10⁴~3.4×10⁴, 4.2×10⁴~4.6×10⁴, 6.4×10⁴~6.8×10⁴, 且均具有活性, 测定PW₁₂和SiW₁₂对酪氨酸酶的抑制效果, 并结合酶动力学法研究其抑制机理. 结果表明, 当PW₁₂和SiW₁₂浓度分别达到13和25 mmol/L时, 酪氨酸酶的活力完全被抑制, 即PW₁₂和SiW₁₂对酪氨酸酶二酚酶具有不同程度的抑制效果. 当所加酶量为0.0173 mg/mL时, PW₁₂和SiW₁₂对酪氨酸酶二酚酶活力的半抑制率 (IC₅₀)分别为1.57和2.31 mmol/L, 它们对酪氨酸酶二酚酶的抑制均为可逆过程. 其中, PW₁₂对二酚酶的抑制类型为混合型, 其K_I及K_IS分别为0.34和0.43 mmol/L, SiW₁₂对二酚酶的抑制类型表现为竞争型, 其K_I为0.59 mmol/L. 综合考虑IC₅₀值和抑制常数等参数, PW₁₂对酪氨酸酶二酚酶的抑制能力优于SiW₁₂.     

Novel approaches for selenium speciation in foodstuffs and biological specimens: A review

Selenium is an essential element for human health. It has been recognized as an antioxidant and chemopreventive agent in cancer. Selenium is known to develop its biological activity via selenocysteine residue in the catalytically active centre of selenoproteins. The main source of selenium in human beings is the diet. However, in several regions of the world the content of selenium in diet has been estimated insufficient for a correct expression of the proteins. The beneficial effects of selenium on human health are strongly dependent on its chemical form and concentration. This review critically evaluated the state-of-the art of selenium speciation in biological matrices mainly focused in nutritional and food products. Besides the number of publications related to selenium speciation, isolation and accurate characterization and quantification of selenium species is still a challenge. Hyphenated techniques based on coupling chromatography separation with inductively coupled plasma spectrometry (ICP-MS) and its combination with molecular mass spectrometry (ESI-MS, ESI-MS-MS and MALDI-TOF) and isotopic dilution allow identification, quantification and structural characterization of selenium species. Particular attention is paid in the development of Se-enriched food and nutritional products and how the application of the techniques mentioned above is mandatory to get reliable results on selenium metabolisms in these particular matrices.     

Improved consistency in 2D gel electrophoresis: Sheep plasma as a test case

Two‐dimensional (2D) gel electrophoresis is a well‐proven proteomic technique; however, sample‐specific optimisation can often be necessary in order to get consistent quantitation. In particular, plasma samples are often smeared on 2D gels making spot matching difficult. A variety of different sample preparation and 2D methods were tested by using sheep plasma, and it was found that lowering sample pH prior to precipitation, using a long voltage gradient for isoelectric focusing and the inclusion of carrier ampholytes in the electrode wicks, improved both the quality and consistency of spot resolution. Analysis of the internal standards from two different DIGE experiments, one with conventional methodology and one with the improved method, showed that along with substantially improving the number of spots resolved, the average CV (coefficient of variation) of matched standards was lower with the new method. 428 matched spots were found using the improved method compared to 208 matched spots using conventional methodology. For the 174 spots that were matched between the two DIGE experiments, the average CV's of spot volumes were also significantly lower, at 0.20 for the new method compared to 0.24 for the conventional method (p < 0.001).