Multi-point, multi-wavelength fluorescence monitoring of DNA separation in a lab-on-a-chip with monolithically integrated femtosecond-laser-written waveguides

Electrophoretic separation of fluorescently labeled DNA molecules in on-chip microfluidic channels was monitored by integrated waveguide arrays, with simultaneous spatial and wavelength resolution. This is an important step toward point-of-care diagnostics with multiplexed DNA assays

Antigen-specific electrophoretic cell separation for immunological investigations

Preincubation of human blood lymphocytes with cell surface antigen specific antibodies under non-capping conditions reduces the electrophoretic mobility of the corresponding lymphocyte subpopulation. Antigen-positive and antigen-negative cells can be separated by free flow electrophoresis with high yield, purity and viability. The use of fluorescence-labelled second antibodies augments the induced decrease in net surface charge density, and allows rapid detection of antigen-positive cells in the fractions of electrophoresis. Carrier-free cell electrophoresis of human peripheral blood lymphocytes after reaction with anti-IgM-antibody or the monoclonal antibodies OKT4 or OKT8, and sandwich staining with tetrarhodamine isothiocyanate-labelled anti-IgG resulted in the large-scale separation of high pure human B and T lymphocyte subpopulations. Their functional integrity was shown in assays of lymphocyte transformation and of antigen-specific induction and regulation of antibody synthesis in vitro. These separate lymphocyte subpopulations are useful tools for immunological investigations. While, for instance, the effects of drugs on human lymphocytes are obscured by coincident changes in cell composition of the peripheral blood tested that do not by themselves reflect whole body immunocompetence, the cell separation and in vitro assays at a defined cell number and cell composition allow the recording of quantitative changes in the function of different cell subpopulations. We studied the influence of the anesthetic thiopental on separated human lymphocyte subsets. In both polyclonal lectin stimulation and in vitro antibody production, thiopental exhibited a noncytotoxic suppression of lymphocyte functions. B-Cells, T-helper and T-suppressor cells were equally affected and showed the same dose response.(ABSTRACT TRUNCATED AT 250 WORDS

Microchip electrophoresis bioanalytical applications

Microchip electrophoresis (MCE) is a novel analytical technique resulting from miniaturization of capillary electrophoresis (CE) to a planar microfabricated separation device. The consequences of the transfer of CE to MCE in terms of benefits and drawbacks have been identified and commented. The strategies developed to overcome the unfavourable features of the chip with respect to the capillary are briefly described. A method for simultaneous separation of catecholamines and their cationic metabolites has been developed on the microchip. The addition of three modifiers was required to resolve all analytes. The sensitivity of on-chip amperometric detection has been improved by employing an enzyme-catalyzed reaction on the amperometric electrode, as well as by using a carbon nanotube-modified electrode. The developed analytical methodology has been successfully applied for a direct on-chip determination of catecholamines and their metabolites in a mouse brain homogenate. The feasibility of performing affinity measurements as well as isoelectric focusing on the microchip has been demonstrated and available applications of these two electrophoretic modes on a chip have been reviewed. A commercial Shimadzu microchip station has for the first time been applied for high-throughput microchip isoelectric focusing of therapeutic proteins and obtained results have been compared to conventional capillary isoelectric focusing

Functional analysis of human alpha 1(I) procollagen gene promoter. Differential activity in collagen-producing and -nonproducing cells and response to transforming growth factor beta 1.

To gain a further understanding of the regulation of human type I collagen gene expression under physiologic and pathologic conditions, we characterized 5.3 kilobase pairs (kb) of the human alpha 1(I) procollagen gene promoter. A series of deletion constructs containing portions of the alpha 1(I) procollagen 5\u27-flanking region (with end points from -5.3 kb to -84 base pairs (bp)) ligated to the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into NIH/3T3 cells. Maximal CAT activity was observed with constructs having 5\u27 end points from -804 to -174 bp. A further 5\u27 deletion to -84 bp caused a marked reduction in CAT activity. Cells transfected with plasmids containing longer 5\u27-flanking fragments of the alpha 1(I) procollagen gene (-2.3 or -5.3 kb) showed reduced CAT activity compared with the -804 bp construct. The activity of the alpha 1(I) procollagen promoter was much lower in cells that do not normally express type I collagen (HeLa cells) compared with collagen-producing NIH/3T3 cells. The CAT activity of deletion constructs containing longer 5\u27 regions than -84 bp was increased by approximately 2-fold in NIH/3T3 cells treated with transforming growth factor beta 1 (TGF beta 1). Electrophoretic mobility shift assays indicated that protein-DNA complex formation with a probe corresponding to the -170 to -80 bp fragment of the alpha 1(I) procollagen promoter was markedly enhanced in nuclear extracts prepared from TGF beta 1-treated fibroblasts as compared with untreated fibroblasts. The DNA binding activity stimulated by TGF beta 1 was specific for an Sp1-like sequence at positions -164 to -142 bp in the promoter. These results demonstrate that 1) there are both positive and negative cis-acting regulatory elements in the human alpha 1(I) procollagen promoter, 2) these regulatory regions function differently in collagen-producing and -nonproducing cells, 3) the alpha 1(I) procollagen promoter contains TGF beta 1-responsive sequences located between -174 and -84 bp from the transcription start site, and 4) TGF beta 1 caused marked stimulation of the DNA binding activity of a nuclear factor interacting with an Sp1-like binding site located within a region encompassing -164 to -142 bp of the alpha 1(I) procollagen promoter

Regulation of Topoisomerase IIa expression in humans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand

In mammalian cells, the loss or down-regulation of tumour-suppressor genes and/or the mutation or overexpression of proto-oncogenes, whose products promote unregulated proliferation in cells, characterise the process of malignant transformation. This generates mitogenic signals that promote abnormal cell growth resulting in tumour progression. Topoisomerase IIα (topo IIα) is an enzyme present in elevated concentrations in highly proliferating cells due to the requirement for untwisting and unknotting of the DNA which is essential for replication. Because of this requirement, a number of anti-cancer drugs have been designed with topo IIα as their primary target. The effectiveness of these drugs however is limited by the development of resistance. One factor linked to drug resistance is the down-regulation of topo IIα at the transcription level. Expression of topo IIα appears to be regulated through various transcription factors with members of the Spl family having a major contribution. Previous work has shown down regulation of topo IIα can occur at the level of transcription. Nucleotide sequencing of the topo IIα promoter in drug-resistant cell lines has not revealed any mutations thus far. Three known proteins and one uncharacterised protein are capable of interacting with the proximal topo IIα promoter region. The uncharacterised protein may act as a co-activator or a co-repressor depending on the complement of transcription factors associated with the DNA in this region. Because drug resistant cell lines showed modulated expression of these transcription factors, it is important to identify the unknown protein and characterise its role in regulating topo IIα expression. This research aimed to identify the minimal binding site and DNA elements required for the uncharacterised protein to bind, as well as introduce mutations into this proximal region and examine their functional significance. The results of this study could provide insights into the molecular mechanisms responsible for the development of drug resistance, contributing to more efficient and effective methods for the treatment of cancer

In vitro toxicity of nanoceria: effect of coating and stability in biofluids

Due to the increasing use of nanometric cerium oxide in applications, concerns about the toxicity of these particles have been raised and have resulted in a large number of investigations. We report here on the interactions between 7 nm anionically charged cerium oxide particles and living mammalian cells. By a modification of the particle coating including low-molecular weight ligands and polymers, two generic behaviors are compared: particles coated with citrate ions that precipitate in biofluids and particles coated with poly(acrylic acid) that are stable and remain nanometric. We find that nanoceria covered with both coating agents are taken up by mouse fibroblasts and localized into membrane-bound compartments. However, flow cytometry and electron microscopy reveal that as a result of their precipitation, citrate-coated particles interact more strongly with cells. At cerium concentration above 1 mM, only citrate-coated nanoceria (and not particles coated with poly(acrylic acid)) display toxicity and moderate genotoxicity. The results demonstrate that the control of the surface chemistry of the particles and its ability to prevent aggregation can affect the toxicity of nanomaterials.Comment: 33 pages 10 figures, accepted at Nanotoxicolog

Transcriptional regulation of human topoisomerase II beta : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry

Pages 24 and 25 are missing from the original copy.Topoisomerase II has an essential role in maintaining the DNA in the correct topological state required for various cellular processes. Its mechanism of action involves the introduction of a double-Dstranded break into the DNA, passage of a different piece of DNA through the break, followed by the religation of the DNA. Topoisomerase II, in humans, exists as two different isoforms: topoisomerase II alpha, which is cell cycle-Dregulated and highly expressed in rapidly proliferating cells, and topoisomerase II beta, which is ubiquitously expressed and it is not under the influence of the cell cycle. Several chemotherapeutic drugs have been designed to interfere with the catalytic mechanism of the topoisomerase II enzyme. By either stabilising the DNA cleavage complex or interfering with another step of the mechanism, these topoisomerase II targeted drugs promote the entry of the cell into cell death pathways. An increasing problem in the treatment of cancer with these drugs is the rising number of patients with inherited or developed drug-resistance. It has been shown that drug-resistance, at least in part, results from the down-regulation of topoisomerase II expression. The expression of a gene is a highly regulated process and the initiation of transcription represents a major point of regulation. Prior to this study little was known regarding the regulation of transcription of topoisomerase II beta. Understanding the processes surrounding the regulation of this enzyme would provide some insight as to how it is down regulated in drug-resistance. The focus of this study was to examine the role of three elements in the topoisomerase II beta promoter, GCI, ICB1, and ICB2 and the transcription factors that bind to them. Electrophoretic mobility shifts assays revealed that Sp1, Sp3, NF-Y and two uncharacterised proteins are capable of binding to the promoter in vitro. Transient transfection assays showed in vivo that Sp1 was able to activate transcription and that Sp3 inhibited transcription driven by the topoisomerase II beta promoter. In addition the key activating elements appear to be ICB2 and GC1, while ICB1 is inhibitory

Highly specific PCR-RFLP assays for karyotyping the widespread 2Rb inversion in malaria vectors of the Anopheles gambiae complex

Background: Chromosomal inversion polymorphisms play a role in adaptation to heterogeneous environments. Inversion polymorphisms are implicated in the very high ecological flexibility of the three main malaria vector species of the Afrotropical Anopheles gambiae complex, facilitating the exploitation of anthropogenic environmental modifications and promoting a strong association with humans. In addition to extending the species' spatial and temporal distribution, inversions are associated with epidemiologically relevant mosquito behavior and physiology, underscoring their medical importance. We here present novel PCR-RFLP based assays strongly predictive of genotype for the cosmopolitan 2Rb inversion in An. coluzzii and An. gambiae, a development which overcomes the numerous constraints inherent to traditional cytological karyotyping. Methods: We designed PCR-RFLP genotyping assays based on tag SNPs previously computationally identified as strongly predictive (> 95%) of 2Rb genotype. We targeted those tags whose alternative allelic states destroyed or created the recognition site of a commercially available restriction enzyme, and designed assays with distinctive cleavage profiles for each inversion genotype. The assays were validated on 251 An. coluzzii and 451 An. gambiae cytologically karyotyped specimens from nine countries across Africa and one An. coluzzii laboratory colony. Results: For three tag SNPs, PCR-RFLP assays (denoted DraIII, MspAI, and TatI) reliably produced robust amplicons and clearly distinguishable electrophoretic profiles for all three inversion genotypes. Results obtained with the DraIII assay are ≥ 95% concordant with cytogenetic assignments in both species, while MspAI and TatI assays produce patterns highly concordant with cytogenetic assignments only in An. coluzzii or An. gambiae, respectively. Joint application of species-appropriate pairs of assays increased the concordance levels to > 99% in An. coluzzii and 98% in An. gambiae. Potential sources of discordance (e.g. imperfect association between tag and inversion, allelic dropout, additional polymorphisms in the restriction target site, incomplete or failed restriction digestion) are discussed. Conclusions: The availability of highly specific, cost effective and accessible molecular assays for genotyping 2Rb in An. gambiae and An. coluzzii allows karyotyping of both sexes and all developmental stages. These novel tools will accelerate deeper investigations into the role of this ecologically and epidemiologically important chromosomal inversion in vector biology.[Figure not available: see fulltext.