Surfactant-driven optimization of iron-based nanoparticle synthesis: a study on magnetic hyperthermia and endothelial cell uptake.

This work examines the effect of changing the ratio of different surfactants in single-core iron-based nanoparticles with respect to their specific absorption rate in the context of magnetic hyperthermia and cellular uptake by human umbilical vein endothelial cells (HUVEC). Three types of magnetic nanoparticles were synthesized by separately adding oleic acid or oleylamine or a mixture of both (oleic acid/oleylamine) as surfactants. A carefully controlled thermal decomposition synthesis process led to monodispersed nanoparticles with a narrow size distribution. Spherical-shaped nanoparticles were mainly obtained for those synthesized with oleic acid, while the shape changed upon adding oleylamine. The combined use of oleic acid and oleylamine as surfactants in single-core iron-based nanoparticles resulted in a substantial saturation magnetization, reaching up to 140 A m2 kg-1 at room temperature. The interplay between these surfactants played a crucial role in achieving this high magnetic saturation. By modifying the surface of the magnetic nanoparticles using a mixture of two surfactants, the magnetic fluid hyperthermia heating rate was significantly improved compared to using a single surfactant type. This improvement can be attributed to the larger effective anisotropy achieved through the modification with both (oleic acid/oleylamine). The mixture of surfactants enhances the control of interparticle distance and influences the strength of dipolar interactions, ultimately leading to enhanced heating efficiency. Functionalization of the oleic acid-coated nanoparticles with trimethoxysilane results in the formation of a core-shell structure Fe@Fe3O4, showing exchange bias (EB) associated with the exchange anisotropy between the shell and the core. The biomedical relevance of our synthesized Fe@Fe3O4 nanoparticles was demonstrated by their efficient uptake by human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner. This remarkable cellular uptake highlights the potential of these nanoparticles in biomedical applications.

Differences in the pattern of X-linked gene expression between fetal bovine muscle and fibroblast cultures derived from the same muscle biopsies.

The sex determination system in mammals creates an imbalance between males and females in the number of X chromosomes. This imbalance is compensated through transcriptional silencing of one of the two X chromosomes in female diploid cells by epigenetic modifications. Although common for mammals, X inactivation shows marked species-specific differences in mechanisms and end results, and provides a unique opportunity to study epigenetic regulation of gene expression. The aim of the present study was to establish the expression pattern of selected X-linked genes in bovine fetal muscle tissue and muscle fibroblast cultures in order to follow possible modifications at the transcriptional level attributable to in vitro culture. We used heterologous cDNA microarray hybridization and quantitative real-time PCR to study the pattern of expression of X-linked genes including SLC25A6, GAB3, MECP2, RPS4X, JARID1C, UBE1, BIRC4 and SLC16A2. Quantitative real-time PCR analysis in fetal bovine muscle showed higher transcript levels in females for all X-linked genes tested with the exception of SLC25A6, with differences being significant for RPS4X, JARID1C and UBE1. The expression in fibroblast cultures derived from the same samples differed, with significantly higher levels for UBE1, GAB3 and BIRC4, while the rest of the panel of X-linked genes remained unchanged. The changed expression pattern in vitro, probably reflecting modifications in the epigenetic mechanisms that regulate transcriptional activity and gene silencing in X inactivation, has important implications for the advancement of new biotechnologies such as somatic cell nuclear transfer and stem cell therapy.

Detection of mitochondrial single nucleotide polymorphisms using a primer elongation reaction on oligonucleotide microarrays.

We have developed a novel allele-specific primer elongation protocol using a DNA polymerase on oligonucleotide chips. Oligonucleotide primers carrying polymorphic sites at their free 3'end were covalently bound to glass slides. The generation of single-stranded targets of genomic DNA containing single nuclotide polymorphisms (SNPs) to be typed was achieved by an asymmetric PCR reaction or exonuclease treatment of phosphothioate (PTO)-modified PCR products. In the presence of DNA polymerase and all four dNTPs, with Cy3-dUTP replacing dTTP, allele-specific extension of the immobilized primers took place along a stretch of target DNA sequence. The yield of elongated products was increased by repeated reaction cycles. We performed multiplexed assays with many small DNA targets, or used single targets of up to 4.4 kb mitochondrial DNA (mtDNA) sequence to detect multiple SNPs in one reaction. The latter approach greatly simplifies preamplification of SNP-containing regions, thereby providing a framework for typing hundreds of mtDNA polymorphisms.

X chromosome-specific cDNA arrays: identification of genes that escape from X-inactivation and other applications.

Mutant alleles are frequently characterized by low expression levels. Therefore, cDNA array-based gene expression profiling may be a promising strategy for identifying gene defects underlying monogenic disorders. To study the potential of this approach, we have generated an X chromosome-specific microarray carrying 2423 cloned cDNA fragments, which represent up to 1317 different X-chromosomal genes. As a prelude to testing cell lines from patients with X-linked disorders, this array was used as a hybridization probe to compare gene expression profiles in lymphoblastoid cell lines from normal males, females and individuals with supernumerary X chromosomes. Measurable hybridization signals were obtained for more than half of the genes represented on the chip. A total of 53 genes showed elevated expression levels in cells with multiple X chromosomes and many of these were found to escape X-inactivation. Moreover, the detection of a male-viable deletion encompassing three genes illustrates the utility of this array for the identification of small unbalanced chromosome rearrangements.

Patterns of desmocollin synthesis in human epithelia: immunolocalization of desmocollins 1 and 3 in special epithelia and in cultured cells.

Murine monoclonal antibodies (mAbs) which specifically react, in immunoblot and immunolocalization experiments, with the human desmosomal cadherins, desmocollins Dsc1 (mAb Dsc1-U100) and Dsc3 (mAb Dsc3-U114), have allowed to study systematically the synthesis of these proteins in tissues and cultured cells. Application of these mAbs in immunofluorescence microscopy on human skin has shown the presence of Dsc1 in the suprabasal layers of interfollicular epidermis and a specific cell layer of the hair follicle root sheath, whereas Dsc3 has been identified in all living epidermal layers as well as in glandular ducts and in basal matrix cells and the outer root sheath of hair follicles. Dsc3, but not Dsc1, it also present in desmosomes of the basal as well as suprabasal cell layers of other stratified epithelia such as vagina, tongue and esophagus as well as in cells of the basal layer of bladder urothelium and the complex epithelium of trachea. All the diverse one-layered ("simple") epithelia examined were as negative for both, Dsc1 and Dsc3, as were the non-epithelial desmosomes of the intercalated disks of the myocardium. A special situation has been discovered in the thymus. Here the usually single-layered cells of the thymic reticular epithelium are connected by Dsc3-possessing desmosomes, as they also contain typical (type I) hemidesmosomes, whereas Dsc1 is only detected in the "Hassall bodies", spheroidal formations of densely packed reticulum-derived cells which also produce cytokeratins 1 and 10, indicative of suprabasal epidermal differentiation. In cell cultures most, probably all, desmosomes of diverse cell lines derived from stratified squamous epithelia or squamous cell carcinomas, including primary keratinocytes, HaCaT keratinocytes and A-431 carcinoma cells, contain Dsc3. By contrast, Dsc1 has only been detected in local piles of keratinocytes that appear to be in the process of suprabasal differentiation. The antibodies have also allowed to demonstrate that desmosomes of cell lines can contain more than one desmocollin isoform. The observations made by immunofluorescence microscopy are compared with results obtained by in situ hybridization of mRNAs, and the potential value of these mAbs in histology and pathology is discussed.

The widespread human desmocollin Dsc2 and tissue-specific patterns of synthesis of various desmocollin subtypes.

By comparison of the cDNA-derived amino acid sequences and the cell type-specific patterns of synthesis we have identified desmocollin Dsc2 as the most widespread, perhaps ubiquitous desmocollin subtype. Using Northern blot analyses and ribonuclease protection assays we have found an approximately 5.6 kb mRNA encoding Dsc2 in all the diverse human tissues, tumors and cell lines examined that are known to possess desmosomes, i.e. not only epithelial cells but also myocardiac cells and lymph nodes. By contrast, desmocollin subtypes Dsc1 and Dsc3 have been detected only in certain stratified squamous epithelia, with the most conspicuous restriction of Dsc1 to epidermis and--remarkably, but unexplained--lymph nodes, and in certain carcinomas and cell lines derived therefrom. We have also determined that both Dsc2 mRNA splice forms, the one encoding the larger polypeptide a and the one coding for the shorter Dsc2b, occur in all the diverse tissues and cell lines examined. We also show that certain cells such as the epidermal keratinocyte line HaCaT and the vulvar carcinoma-derived line A-431 continually synthesize more than one Dsc subtype. The cell type-specific patterns of synthesis of the various Dsg and Dsc subtypes are discussed in relation to tissue development during embryogenesis and to malignant transformations, and the utilization of reagents for the specific Dsg and Dsc subtypes in tumor diagnosis is proposed.

Desmosomes and cytoskeletal architecture in epithelial differentiation: cell type-specific plaque components and intermediate filament anchorage.

Among the diverse kinds of intercellular, plaque-bearing, cadherin-containing junctions, desmosomes (maculae adhaerentes) represent a major type characterized by the presence of specific transmembrane glycoproteins, i.e. desmosomal cadherins of the desmoglein and desmocollin categories, and the cytoplasmic plaque proteins, desmoplakin I and plakoglobin. Recent studies, however, have shown that the composition of desmosomes is not identical in the various normal and tumorous desmosome-forming tissues and cell cultures, including diverse forms of epithelia and carcinomas, meningothelia and meningiomas, myocardium and the lymph node follicle reticulum. Desmosomes can differ in their specific complement of desmogleins, Dsg1-3, and desmocollins, Dsc1a-3b, as well as in the additional presence and in their relative amounts of certain accessory plaque proteins such as desmoplakin II and plakophilin 1, a basic member of the larger plakoglobin family of proteins ("band 6 protein"). Assembly and function of desmosomes are effected by the interaction of the specific complement of desmosomal cadherins with certain cytoplasmic proteins. In particular, the cytoplasmic portions ("tails") of the desmosomal cadherins contain certain domains and amino acid sequence motifs, identified by mutagenesis and transfection assays, that are essential elements in desmosome formation, notably the assembly of plaque proteins, and in the site-specific anchorage of intermediate-sized filaments (IFs) of the cytoskeleton, thereby contributing to the specific intracellular as well as supracellular, i.e. tissue, architecture.

Desmosomes--dual junctional principles of intra- and supracellular order in epithelial differentiation and tissue formation.

The cells of most normal and malignantly growing tissues are connected by "adhering junctions", i.e. distinct sites of "homotypic" contact between the plasma membranes of two cells of the same or a similar kind, associated on the cytoplasmic side by a dense plaque at which often bundles of cytoskeletal filaments anchor. Of the various types of adhering junctions desmosomes are characteristic of epithelia and carcinomas but also occur in some other cell types. Their molecular components have recently been identified and characterized by cDNA-cloning and sequencing. Unexpectedly, the molecular complement of desmosomes has been found to show certain differences in different epithelia, with particularly complex patterns in stratified squamous epithelia as well as in tumors and cultured cell lines derived therefrom. In addition, molecular principles important in the assembly of desmosomes and in the specific anchorage of intermediate-sized filaments (IFs) at desmosomal plaques have been elucidated. The possible value of cell type-specific isoforms of desmosomal components as markers for the subtyping of carcinomas and the role of desmosomal cadherins during invasion and metastasis of carcinomas are discussed.