Tracking the pathway of calcium phosphate/DNA nanoparticles during cell transfection by incorporation of red-fluorescing tetramethylrhodamine isothiocyanate–bovine serum albumin into these nanoparticles

Calcium phosphate nanoparticles were prepared by precipitation from water and were then functionalized by DNA. These particles are taken up by living cells and function as gene transfer agents, i.e., the DNA is brought into a cell’s nucleus and is incorporated there into the cell’s genome (transfection). DNA which encodes for enhanced green fluorescent protein leads to green fluorescence of successfully transfected cells. By adding the red-fluorescing marker tetramethylrhodamine isothiocyanate–bovine serum albumin (TRITC-BSA) to the nanoparticles, their pathway into the cell and within the cell could be followed by fluorescence microscopy. A clear correlation between the uptake of nanoparticles and the efficiency of transfection was found. Aggregates of DNA/TRITC-BSA alone were not able to enter the cells, i.e., the inorganic nanoparticles are necessary as a carrier through the cell membrane.     

Thermodynamic Characterization of ppGpp Binding to EF-G or IF2 and of Initiator tRNA Binding to Free IF2 in the Presence of GDP, GTP, or ppGpp

In addition to their natural substrates GDP and GTP, the bacterial translational GTPases initiation factor (IF) 2 and elongation factor G (EF-G) interact with the alarmone molecule guanosine tetraphosphate (ppGpp), which leads to GTPase inhibition. We have used isothermal titration calorimetry to determine the affinities of ppGpp for IF2 and EF-G at a temperature interval of 5-25 °C. We find that ppGpp has a higher affinity for IF2 than for EF-G (1.7-2.8 μM K_dversus 9.1-13.9 μM K_d at 10-25 °C), suggesting that during stringent response in vivo, IF2 is more responsive to ppGpp than to EF-G. We investigated the effects of ppGpp, GDP, and GTP on IF2 interactions with fMet-tRNA^fMet demonstrating that IF2 binds to initiator tRNA with submicromolar K_d and that affinity is altered by the G nucleotides only slightly. This—in conjunction with earlier reports on IF2 interactions with fMet-tRNA^fMet in the context of the 30S initiation complex, where ppGpp was suggested to strongly inhibit fMet-tRNA^fMet binding and GTP was suggested to strongly promote fMet-tRNA^fMet binding—sheds new light on the mechanisms of the G-nucleotide-regulated fMet-tRNA^fMet selection.     

Cks1 is required for tumor cell proliferation but not sufficient to induce hematopoietic malignancies

The Cks1 component of the SCF(Skp2) complex is necessary for p27(Kip1) ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27(Kip1) levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27(Kip1) levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27(Kip1). To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.     

Correlated cleavage of single- and double-stranded substrates by uracil-DNA glycosylase

Uracil-DNA glycosylase (Ung) can quickly locate uracil bases in an excess of undamaged DNA. DNA glycosylases may use diffusion along DNA to facilitate lesion search, resulting in processivity, the ability of glycosylases to excise closely spaced lesions without dissociating from DNA. We propose a new assay for correlated cleavage and analyze the processivity of Ung. Ung conducted correlated cleavage on double- and single-stranded substrates; the correlation declined with increasing salt concentration. Proteins in cell extracts also decreased Ung processivity. The correlated cleavage was reduced by nicks in DNA, suggesting the intact phosphodiester backbone is important for Ung processivity.     

Determination of the Activity of Immobilized Biocatalysts in the Case of Poorly Soluble Substrates: The Example of D-5Phenylhydantoinase

The present communication describes the determination of activity of immobilized biocatalysts, from progress curves, in the case of a poorly soluble substrate. Computer simulation is used for the comparison of five well known methods for the determination of initial velocity of the reaction with the modified method of smoothing experimental data by cubic spline. The computer simulation data show that, in the case of poorly soluble substrate, it is expedient to use the method of data linearization in the co-ordinate system p/t versus p and method of splines.     

Micropropagation of the endangered species Malus niedzwetzkyana for conservation biodiversity in Kazakhstan

In Vitro Cellular & Developmental Biology - Plant - Biodiversity conservation requires advanced and effective ex situ plant propagation techniques. The present study was conducted to optimize...     

Quantitative dissection of color patterning in the foliar ornamental coleus

Coleus (Coleus scutellarioides) is a popular ornamental plant that exhibits a diverse array of foliar color patterns. New cultivars are currently hand selected by both amateur and experienced plant breeders. In this study, we reimagine breeding for color patterning using a quantitative color analysis framework. Despite impressive advances in high-throughput data collection and processing, complex color patterns remain challenging to extract from image datasets. Using a phenotyping approach called “ColourQuant,” we extract and analyze pigmentation patterns from one of the largest coleus breeding populations in the world. Working with this massive dataset, we can analyze quantitative relationships between maternal plants and their progeny, identify features that underlie breeder-selections, and collect and compare public input on trait preferences. This study is one of the most comprehensive explorations into complex color patterning in plant biology and provides insights and tools for exploring the color pallet of the plant kingdom.

Quantitative analysis of color patterning in a large coleus breeding population reveals color features that are associated with aesthetic value.     

Polyfluorinated salicylic acid derivatives as analogs of known drugs: Synthesis,molecular docking and biological evaluation

We have developed the convenient methods for synthesis of polyfluorosalicylic acids and their derivatives. For the first time the biological properties of polyfluorosalicylates were investigated in vitro (permeability through the biological membranes, COX-1 inhibitory action) and in vivo (anti-inflammatory, analgesic activities, acute toxicity). Molecular docking of polyfluorinated salicylates confirmed in vitro and in vivo experiments.     

Heterometallic complex formation on p-sulfonatothiacalix[4]arene platform resulting in pH- and redox-modification of [Ru(bpy)3] luminescence

The pH-dependent heterometallic complex formation with p-sulfonatothiacalix[4]arene (TCAS) as bridging ligand in aqueous solutions was revealed by the use of spectrophotometry, nuclear magnetic relaxation and fluorimetry methods. The novelty of the structural motif presented is that the appendance of emission metal center ([Ru(bpy)₃]²⁺) is achieved through the cooperative non-covalent interactions with the upper rim of TCAS. The second metal block (Fe(III), Fe(II) and Mn(II)), bound with the lower rim of TCAS in the inner sphere coordination mode is serving as quencher of [Ru(bpy)₃]²⁺ emission. The difference between the complex ability of Fe(III) and Fe(II) ions provides pH conditions for redox-dependent emission of [Ru(bpy)₃]²⁺.