Electrophoretic mobility of cell nuclei (EMN index) as a biomarker of the biological aging process: Considering the association between EMN index and age

The present study examined whether a specific property of cell microstructures may be useful as a biomarker of aging. Specifically, the association between age and changes of cellular structures reflected in electrophoretic mobility of cell nuclei index (EMN index) values across the adult lifespan was examined. This report considers findings from cross sections of females (n = 1273) aged 18–98 years, and males (n = 506) aged 19–93 years. A Biotest apparatus was used to perform intracellular microelectrophoresis on buccal epithelial cells collected from each individual. EMN index was calculated on the basis of the number of epithelial cells with mobile nuclei in reference to the cells with immobile nuclei per 100 cells. Regression analyses indicated a significant negative association between EMN index value and age for men (r = −0.71, p < 0.001) and women (r = −0.60, p < 0.001); demonstrating a key requirement that must be met by a biomarker of aging. The strength of association observed between EMN index and age for both men and women was encouraging and supports the potential use of EMN index for determining a biological age of an individual (or a group). In this study, a new attempt of complex explanation of cellular mechanisms contributing to age related changes of the EMN index was made. In this study, a new attempt of complex explanation of cellular mechanisms contributing to age related changes of the EMN index was made. EMN index has demonstrated potential to meet criteria proposed for biomarkers of aging and further investigations are necessary.

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C-12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150 000 plates/meter) and good reproducibility (migration time with RSD < 0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.

Size, electrophoretic mobility, and ion dissociation of vesicles prepared with synthetic amphiphiles

Vesicles prepared with synthetic amphiphiles (dioctadecyldimethylammonium bromide and chloride, dihexadecyl phosphate and its sodium salt) were obtained by sonication, ethanol injections, and chloroform injections. The hydrodynamic diameter of vesicles (Dh), estimated from the diffusivity measured by quasielastic light scattering, ranged from 230 to 3000 Å. The electrophoretic mobility (Um) was measured by free-flow electrophoresis. The zeta potential (ζ) and the degree of counterion dissociation (α) of the vesicles were calculated from Um and conductivity data, α decreased with increasing Dh of the vesicles, probably due to the decreasing headgroup area and the increasing counterion association needed to relax the surface electrostatic potential. The electrophoretic mobility was also calculated (Uc) according to an impenetrable, nonconducting sphere model with a spherically symmetric charge distribution approximation. Within the limits of the experimental error(s) of the (different) methods employed and the assumptions made in the calculations, the fact that the Um/Uc ratio ranged from 1.3 to 7.5 was considered to be a good agreement between the calculated and the experimental values. © 1990 American Chemical Society.

The leucine zipper may induce electrophoretic mobility anomalies without DNA bending

Numerous proteins bend DNA upon binding, a phenomenon of potential significance for regulation of gene expression and chromatin. DNA bending is commonly predicted from the presence of electrophoretic mobility anomalies in protein–DNA complexes. However, as compared with electrophoretic methods, several DNA binding oncoprotein families do not display comparable evidence of DNA bends in x-ray structural studies. Herein, circularization kinetics and affinity measurements with prebent DNA templates were employed to assess bending and DNA structural preferences for Max and other basic helix–loop–helix/leucine zipper proteins. In this way, proteins in the Myc/Max basic helix–loop–helix/leucine zipper family were found not to bend DNA in solution but to actually stabilize DNA in an unbent configuration that resists circularization. The mobility anomaly was found to be induced by the leucine zipper protein motif, rather than structural distortions of DNA. Thus rigid protein domain structures may induce anomalous electrophoretic mobility. Moreover, the energetic preference of non-DNA bending proteins for unbent templates suggests mechanisms whereby chromatin structure may regulate transcription.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear “gems,” along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

Soft-particle model analysis of effect of LPS on electrophoretic softness of Acidithiobacillus ferrooxidans grown in presence of different metal ions

The effect of Surface lipopolysaccharides (LPS) on the electrophoretic softness and fixed charge density in the ion-penetrable layer of Acidithiobacillus ferrooxidans cells grown in presence of copper or arsenic ions have been discussed, The electrophoretic mobility data were analyzed using the soft-particle electrophoresis theory. Cell surface potentials of all the strains based on soft-particle theory were lower than those estimated using the conventional Smoluchowski theory, Exposure to metal ions increased the Surface electrophoretic softness with decrease in the fixed charge density. Effect of cell surface lipopolysaccharides on the model parameters are investigated and discussed.

Comparative electrophoretic patterns of lactase dehydrogenase and malate dehydrogenase in five Lake Victoria cichlid species

Biochemical techniques designed to compare species on the basis of protein differences were started by NUTTALL (1904) who used immunological methods to compare the serum of humans with that of other primates. Since then more refined techniques have led to better results at the protein level in taxonomy, The analyses of proteins are considered to be the simplest indirect approach to understanding the structure and function of the genetic material, deoxyribonucleic acid (DNA). Interest in these analyses arises because of the close relationship between protein structure and gene structure. Thus by comparing the properties of homologous proteins from different taxa one is in essence comparins their genes (GORMAN er al., 1971). It is now an established fact that genetic information coded in molecules of DNA is translated through a series of reactions in the structure of proteins which form the principal morphological units of the animal body at the molecular level of organization (SIBLEY, 1952). A convenient method of comparing molecular differences between species is to measure the electrophoretic mobility of proteins in a starch gel medium (ASPINWALL and TSUYUKI, 1968) or acrylamide gel (RAYMOND and WEINTRAUB, 1959; BOUCK and BALL, 1968). Proteins with enzymatic properties can be compared on the basis of catalytic activity in the presence or absence of inhibitors (KAPLAN et al., 1959); BAILEY et al., t 1970). A combination of gel electrophoresis and histochemical enzyme detection techniques (HUNTER and MARKERT, 1957) makes it possible to combine electrophoretic mobility anti catalytic activity comparison, Enzyme patterns exhibited in starch gel or acrylamide gel have been used to classify different species. BOUCK and BALL (1968)working with lactate dehydrogenase in species of Trout found that each Trout species had LDH pattern characterbtic of that species. ASPINIWALL and TSUYUKI (1968) used muscle protein electrophoretic patterns to identify hybrid fishes. TSUYUKI and ROBERTS (1963) and TSUYUKI et al. (1964-65) found that myogen protein patterns in fishes were species specific. The myogen patterns within one family were remarkably parallel with the existing morphometric classification and these patterns constituted a single criterion by which the fishes could be identified. The fish used in these investigations were collected from shallow waters (10 metres) of Lake Victoria in two areas, Jinja and Kisumu, using gillnets and beach-seines. The study included ten specimens of each of the following specIes: (l) Haplochromis michaeli (2) Haploehromis obems (3) Astatoreochromis ulluaudi (4) Tilapia zillii and (5) Tilapia nilotica.

Electrophoretic variation in adenylate kinase of Neisseria meningitidis is due to inter- and intraspecies recombination.

In prokaryotic and eukaryotic organisms, the electrophoretic variation in housekeeping enzymes from natural populations is assumed to have arisen by the accumulation of stochastic predominantly neutral mutations. In the naturally transformable bacterium Neisseria meningitidis, we show that variation in the electrophoretic mobility of adenylate kinase is due to inter- and intraspecies recombination rather than mutation. The nucleotide sequences of the adenylate kinase gene (adk) from isolates that express the predominant slow electrophoretic variant were rather uniform, differing in sequence at an average of 1.1% of nucleotide sites. The adk sequences of rare isolates expressing the fast migrating variant were identical to each other but had a striking mosaic structure when compared to the adk genes from strains expressing the predominant variant. Thus the sequence from the fast variants was identical to those of typical slow variants in the first 158 bp of the gene but differed by 8.4% in the rest of the gene (nt 159-636). The fast electrophoretic variant appears to have arisen by the replacement of most of the meningococcal gene with the corresponding region from the adk gene of a closely related Neisseria species. The adk genes expressing the electrophoretic variant with intermediate mobility were perfect, or almost perfect, recombinants between the adk genes expressing the fast and slow variants. Recombination may, therefore, play a major role in the generation of electrophoretically detectable variation in housekeeping enzymes of some bacterial species.

Membrane associated transporter protein gene (SLC45A2) and the genetic basis of normal human pigmentation variation

This work is concerned with the genetic basis of normal human pigmentation variation. Specifically, the role of polymorphisms within the solute carrier family 45 member 2 (SLC45A2 or membrane associated transporter protein; MATP) gene were investigated with respect to variation in hair, skin and eye colour ― both between and within populations. SLC45A2 is an important regulator of melanin production and mutations in the gene underly the most recently identified form of oculocutaneous albinism. There is evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated with normal pigmentation variation between populations. Therefore, the underlying hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or regulation of the protein, thereby altering the important role it plays in melanogenesis and providing a mechanism for normal pigmentation variation. In order to investigate the role that SLC45A2 polymorphisms play in human pigmentation variation, a DNA database was established which collected pigmentation phenotypic information and blood samples of more than 700 individuals. This database was used as the foundation for two association studies outlined in this thesis, the first of which involved genotyping two previously-described non-synonymous polymorphisms, p.Glu272Lys and p.Phe374Leu, in four different population groups. For both polymorphisms, allele frequencies were significantly different between population groups and the 272Lys and 374Leu alleles were strongly associated with black hair, brown eyes and olive skin colour in Caucasians. This was the first report to show that SLC45A2 polymorphisms were associated with normal human intra-population pigmentation variation. The second association study involved genotyping several SLC45A2 promoter polymorphisms to determine if they also played a role in pigmentation variation. Firstly, the transcription start site (TSS), and hence putative proximal promoter region, was identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE). Two alternate TSSs were identified and the putative promoter region was screened for novel polymorphisms using denaturing high performance liquid chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was identified along with other previously described single nucleotide polymorphisms (c.–1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three polymorphisms were associated with skin colour such that the –1721G, +dup and –1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium was observed between the promoter and coding region polymorphisms, suggesting independent effects. The association analyses were complemented with functional data, showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2 transcriptional activity. Based on in silico bioinformatic analysis that showed these alleles remove a microphthalmia-associated transcription factor (MITF) binding site, and that MITF is a known regulator of SLC45A2 (Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation by altering MITF binding affinity. Further characterisation of the SLC45A2 promoter was carried out using luciferase reporter assays to determine the transcriptional activity of different regions of the promoter. Five constructs were designed of increasing length and their promoter activity evaluated. Constitutive promoter activity was observed within the first ~200 bp and promoter activity increased as the construct size increased. The functional impact of the –1721G, +dup and –1169A alleles, which removed a MITF consensus binding site, were assessed using electrophoretic mobility shift assays (EMSA) and expression analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed that the promoter polymorphisms affected DNA-protein binding. Interestingly, however, the protein/s involved were not MITF, or at least MITF was not the protein directly binding to the DNA. In an effort to more thoroughly characterise the functional consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression results suggested a coordinated expression of pigmentation genes. This thesis has substantially contributed to the field of pigmentation by showing that SLC45A2 polymorphisms not only show allele frequency differences between population groups, but also contribute to normal pigmentation variation within a Caucasian population. In addition, promoter polymorphisms have been shown to have functional consequences for SLC45A2 transcription and the expression of other pigmentation genes. Combined, the data presented in this work supports the notion that SLC45A2 is an important contributor to normal pigmentation variation and should be the target of further research to elucidate its role in determining pigmentation phenotypes. Understanding SLC45A2’s function may lead to the development of therapeutic interventions for oculocutaneous albinism and other disorders of pigmentation. It may also help in our understanding of skin cancer susceptibility and evolutionary adaptation to different UV environments, and contribute to the forensic application of pigmentation phenotype prediction.

DNA technology for the detection of common genetic variants that predispose to thrombophilia

With the identification of common single locus point mutations as risk factors for thrombophilia, many DNA testing methodologies have been described for detecting these variations. Traditionally, functional or immunological testing methods have been used to investigate quantitative anticoagulant deficiencies. However, with the emergence of the genetic variations, factor V Leiden, prothrombin 20210 and, to a lesser extent, the methylene tetrahydrofolate reductase (MTHFR677) and factor V HR2 haplotype, traditional testing methodologies have proved to be less useful and instead DNA technology is more commonly employed in diagnostics. This review considers many of the DNA techniques that have proved to be useful in the detection of common genetic variants that predispose to thrombophilia. Techniques involving gel analysis are used to detect the presence or absence of restriction sites, electrophoretic mobility shifts, as in single strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification. Such techniques may be sensitive, but are unwielding and often need to be validated objectively. In order to overcome some of the limitations of gel analysis, especially when dealing with larger sample numbers, many alternative detection formats, such as closed tube systems, microplates and microarrays (minisequencing, real-time polymerase chain reaction, and oligonucleotide ligation assays) have been developed. In addition, many of the emerging technologies take advantage of colourimetric or fluorescence detection (including energy transfer) that allows qualitative and quantitative interpretation of results. With the large variety of DNA technologies available, the choice of methodology will depend on several factors including cost and the need for speed, simplicity and robustness. © 2000 Lippincott Williams & Wilkins.

Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus x domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

The binding of nuclear factors to the as-1 element in the CaMV 35S promoter is affected by cytosine methylation in vitro

Transcriptional gene silencing (TCS) is often associated with an increased level of cytosine methylation in the affected promoters. The effect of methylation of the cauliflower mosaic virus (CaMV) 35S promoter sequence on its binding to factors present in the nuclei was analyzed by electrophoretic mobility shift assays using extracts of petunia flowers. Specific DNA-protein interactions were detected in the region of the CaMV 35S promoter that contains the as-1 element and the region between -345 and -208. The binding of protein factor(s) to the as-1 element was influenced by cytosine methylation, whereas the binding to the region between -345 and -208 was unaffected. The results suggest that cytosine methylation of the as-1 element potentially affects the activity of the CaMV 35S promoter. © Georg Thieme Verlag KG Stuttgart.

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes; Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA, The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a. property similar to the eukaryotic type I topoisomerases, The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.

Left-Handed DNA in Synthetic and Topologically Constrained Form V-DNA

We have investigated structural transitions in Poly(dG-dC) and Poly(dG-Me5dC) in order to understand the exact role of cations in stabilizing left-handed helical structures in specific sequences andthe biological role, if any, of these structures. From a novel temperature dependent transition it has been shown that a minor fluctuation in Na+ concentration at ambient temperature can bring about Β to Ζ transition. Forthe first time, wehave observed a novel double transition in poly(dG-Me5dC) as the Na+ concentration is gradually increased. This suggests that a minor fluctuation in Na+ concentration in conjunction with methylation may transform small stretches of CG sequences from one conformational state to another. These stretches could probably serve as sites for regulation. Supercoiled formV DNA reconstituted from pBR322 and pßG plasmids have been studied as model systems, in order to understand the nature and role of left-handed helical conformation in natural sequences. A large portion of DNA in form V, obtained by reannealing the two complementary singlestranded circles is forced to adopt left-handed double helical structure due to topological constraints (Lk = 0). Binding studies with Z-DNA specific antibody and spectroscopic studies confirm the presence of left-handed Z-structure in the pßG and pßR322 form V DNA. Cobalt hexamine chloride, which induces Z-form in Poly(dG-dC) stabilizes the Z-conformation in form V DNA even in the non-alternating purine-pyrimidine sequences. A reverse effect is observed with ethidium bromide. Interestingly, both topoisomerase I and II (from wheat germ) act effectively on form V DNA to give rise to a species having an electrophoretic mobility on agarose gel similar to that of open circular (form II) DNA. Whether this molecule is formed as a result of the left-handed helical segments of form V DNA undergoing a transition to the right-handed B-form during the topoisomerase action remains to be solved.