Apatite formation on nanomaterial calcium phosphate/poly-DL-lactide-co-glycolide in simulated body fluid

Simulated body fluid (SBF) is an artificial fluid which has ionic composition and ionic concentration similar to human blood plasma. Purpose: This paper compares the interaction between the nanomaterial containing calcium phosphate/poly-dl-lactide-co-glycolide (N-CP/PLGA) and SBF, in order to investigate whether and to what extent inorganic ionic composition of human blood plasma leads to the aforementioned changes in the material. Methods: N-CP/PLGA was incubated for 1, 2, 3, and 5 weeks in SBF. The surface of the material was analyzed on SEM-EDS and FTIR spectrometer, while SBF was subjected to pH and electrical conductivity measurement. Results: Our results indicate that dissolution of the polymer component of the material N-CP/PLGA and precipitation of the material similar to hydroxyapatite on its surface are based on the morphologic changes seen in this material. Conclusions: The mechanism of the apatite formation on the bioceramic surface was intensively studied and was considered crucial in designing the new biomaterials. The results obtained in this work indicate that N-CP/PLGA may be a good candidate for application to bone regeneration.     

A versatile fluorescence-based multiplexing assay for CAPS genotyping on capillary electrophoresis systems

Recent advances in next-generation sequencing techniques and the development of genomics resources for crop plants with large genomes allow the detection of a large number of single nucleotide polymorphisms (SNPs) and their use in a high-throughput manner. However, such large numbers of SNPs are on the one hand not needed in some plant breeding projects and on the other hand not affordable in some cases, raising the need for fast and low-cost innovative techniques for marker detection. In marker selection in plant breeding programs, cleaved amplified polymorphic sequence (CAPS) markers still play a significant role as a complement to other high-throughput methods for SNP genotyping. New methods focusing on the acceleration of CAPS-based genotyping are therefore highly desirable. The combination of the classical CAPS method and a M13-tailed primer multiplexing assay was used to develop an agarose-gel-free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence-labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected in a capillary electrophoresis system. This method allowed the cost-effective genotyping of several SNPs in barley in a multiplexed manner at an overall low cost in a short period of time. This new method was efficiently combined with the simultaneous detection of simple sequence repeats in the same electrophoresis run, resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.     

Identification of agonists and antagonists of the human melanocortin-4 receptor from piperazinebenzylamines

SAR studies of a series of piperazinebenzylamines resulted in identification of potent agonists and antagonists of the human melanocortin-4 receptor. Thus, the 1,2,3,4-tetrahydroisoquinolin-1-ylacetyl compound 12e and the quinolin-3-ylcarbonyl analogue 12l possessed K(i) values of 6.3 and 4.5 nM, respectively. Interestingly, 12e was a full agonist with an EC(50) value of 31 nM, and 12l was a weak partial agonist (IA=17%) and functioned as an antagonist (IC(50)=300 nM).     

Effects of subchronic acrylamide treatment on the endocrine pancreas of juvenile male Wistar rats

Acrylamide (AA) is a well-known industrial monomer with carcinogenic, mutagenic, neurotoxic and endocrine disruptive effects on living organisms. AA has been the subject of renewed interest owing to its presence in various food products. We investigated the potential adverse effects of oral AA treatment on the endocrine pancreas of juvenile rats using histochemical, immunohistochemical, stereological and biochemical methods. Thirty juvenile male Wistar rats were divided into one control and two AA treatment groups: one treated with 25 mg/kg AA and the other treated with 50 mg/kg AA for 21 days. We found a significant decrease in β-cell mass. The significant decrease in β-cell optical density and unchanged blood glucose levels indicate that normoglycemia in AA treated rats may result from intensive exocytosis of insulin-containing secretory granules. By contrast with β-cells, we observed increased α-cell mass. The slight increase in α-cell cytoplasmic volume suggests retention of glucagon in α-cells, which is consistent with the significant increase in α-cell optical density for AA treated animals. The number of islets of Langerhans did not change significantly in AA treated groups. Our findings suggest that AA treatment causes decreased β-cell mass and moderate α-cell mass increase in the islets of Langerhans of juvenile male Wistar rats.     

The energetics of specific binding of AT-hooks from HMGA1 to target DNA

The interaction of the second and third AT-hooks of HMGA1 (formerly HMGI/Y), which bind selectively in the minor groove of an AT-rich DNA sequence, was studied at different temperatures and ionic strengths by spectropolarimetry, spectrofluorimetry, isothermal titration calorimetry and differential scanning calorimetry. The data show that binding of the ten amino acid core element of the two AT-hooks, which penetrates deep into the minor groove, is entropically driven: both the entropy and enthalpy of association of the peptides to the target DNA are positive up to 50 degrees C. The seven amino acid extension of the core in the second AT-hook, which extends out from the minor groove and loops over the phosphodiester backbone, adds a substantial negative enthalpic component into the binding of the 17 residue DBD2 peptide to DNA that corresponds in magnitude to the enthalpy of formation of two hydrogen bonds. The ionic strength dependence of the association constant allowed an estimation of the electrostatic component of binding and, by subtraction, the contribution of the non-electrostatic component, which results from dehydration of the contacting surfaces and makes up almost 70% of the total energy of complex formation. The exceptionally large positive entropy and enthalpy of association of the core AT-hook peptides with target DNA suggest that the water, which is removed from the minor groove of DNA upon binding, is in a highly ordered state. Acetylation of the lysine residue in the second AT-hook, which corresponds to Lys65 of HMGA1, has little effect on the DNA binding; so it appears that repression of the hIFNbeta gene, which follows this modification, is not a direct result of the abrogation of DNA binding.     

Current advances in research of cytochrome c oxidase

The function of cytochrome c oxidase as a biomolecular nanomachine that transforms energy of redox reaction into protonmotive force across a biological membrane has been subject of intense research, debate, and controversy. The structure of the enzyme has been solved for several organisms; however details of its molecular mechanism of proton pumping still remain elusive. Particularly, the identity of the proton pumping site, the key element of the mechanism, is still open to dispute. The pumping mechanism has been for a long time one of the key unsolved issues of bioenergetics and biochemistry, but with the accelerating progress in this field many important details and principles have emerged. Current advances in cytochrome oxidase research are reviewed here, along with a brief discussion of the most complete proton pumping mechanism proposed to date, and a molecular basis for control of its efficiency.     

Prediction of Associations between microRNAs and Gene Expression in Glioma Biology

Despite progress in the determination of miR interactions, their regulatory role in cancer is only beginning to be unraveled. Utilizing gene expression data from 27 glioblastoma samples we found that the mere knowledge of physical interactions between specific mRNAs and miRs can be used to determine associated regulatory interactions, allowing us to identify 626 associated interactions, involving 128 miRs that putatively modulate the expression of 246 mRNAs. Experimentally determining the expression of miRs, we found an over-representation of over(under)-expressed miRs with various predicted mRNA target sequences. Such significantly associated miRs that putatively bind over-expressed genes strongly tend to have binding sites nearby the 3'UTR of the corresponding mRNAs, suggesting that the presence of the miRs near the translation stop site may be a factor in their regulatory ability. Our analysis predicted a significant association between miR-128 and the protein kinase WEE1, which we subsequently validated experimentally by showing that the over-expression of the naturally under-expressed miR-128 in glioma cells resulted in the inhibition of WEE1 in glioblastoma cells.     

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 - one of the ligands of the Cu(B) redox center - which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second "chemical" proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.