Magnetoresistive-based biosensors and biochips

Over the past five years, magnetoelectronics has emerged as a promising new platform technology for biosensor and biochip development. The techniques are based on the detection of the magnetic fringe field of a magnetically labeled biomolecule interacting with a complementary biomolecule bound to a magnetic-field sensor. Magnetoresistive-based sensors, conventionally used as read heads in hard disk drives, have been used in combination with biologically functionalized magnetic labels to demonstrate the detection of molecular recognition. Real-world bio-applications are now being investigated, enabling tailored device design, based on sensor and label characteristics. This detection platform provides a robust, inexpensive sensing technique with high sensitivity and considerable scope for quantitative signal data, enabling magnetoresistive biochips to meet specific diagnostic needs that are not met by existing technologies.     

Glyco-optimization of aminoglycosides: new aminoglycosides as novel anti-infective agents

Glyco-optimization (OPopS) of aminoglycosides has been performed by replacing the existing sugar moiety with a variety of sugar derivatives. Glycosylation of the 6-position of nebramine provided a library of novel 4,6-linked aminoglycosides (AMGs). Among them, compounds 8b,g,i,l, and 8u with 2"-amino, 2",3"-diamino, 2",4"-diamino, 3",4"-diamino, 3"-amino groups, respectively, showed significant antimicrobial activity against Gram-(+) and -(-) bacteria. Several were particularly potent against Pseudomonus aeruginosa with MICs in the 1-2 microg/mL range.     

Synthesis and pharmacological evaluation of prenylated and benzylated pterocarpans against snake venom

Edunol (3), a pterocarpan isolated from Harpalyce brasiliana, a plant used in the northeast of Brazil against snakebites, was obtained by synthesis and showed antimyotoxic, antiproteolytic and PLA2 inhibitor properties. These proprieties could be improved through the synthesis of a bioisoster (5), where the prenyl group was replaced by the benzyl group.     

Data exploration tools for the Gene Ontology database

MOTIVATION: To improve the ability of biologists (both researchers and students) to ask biologically interesting questions of the Gene Ontology (GO) database and to explore the ontologies by seeing large portions of the ontology graphs in context, along with details of individual terms in the ontologies. RESULTS: GoGet and GoView are two new tools built as part of an extensible web application system based on Java 2 Enterprise Edition technology. GoGet has a user interface that enables users to ask biologically interesting questions, such as (1) What are the DNA binding proteins involved in DNA repair, but not in DNA replication? and (2) Of the terms containing the word triphosphatase, which have associated gene products from mouse, but not fruit fly? The results of such queries can be viewed in a collapsed tabular format that eases the burden of getting through large tables of data. GoView enables users to explore the large directed acyclic graph structure of the ontologies in the GO database. The two tools are coordinated, so that results from queries in GoGet can be visualized in GoView in the ontology in which they appear, and explorations started from GoView can request details of gene product associations to appear in a result table in GoGet. AVAILABILITY: Free access to the GoGet query tool and free download of the GoView ontology viewer are provided to all users at http://db.math.macalester.edu/goproject. In addition, source code for the GoView tool is also available from this site, along with a user manual for both tools.     

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.     

Vitamin E or coenzyme Q10 administration is not fully advantageous for heart mitochondrial function in diabetic goto kakizaki rats

The heart is one of the organs affected during the later stages of diabetes. Mitochondrial function has already been proposed to be affected during the course of diabetes. Nevertheless, little information is known concerning the impact of antioxidants in heart mitochondria of a milder model for diabetes, such as the Goto-Kakizaki (GK) rat, where mitochondrial function appears ameliorated. The objective of this work was to test if injections of Vitamin E and Coenzyme Q10, alone and in combination, were able to modify mitochondrial performance in the hearts of GK rats. Several aspects of mitochondrial function were measured, such as the respiratory control ratio and the electric potential, as well as the mitochondrial accumulation of Vitamin E and Coenzymes Q9 and Q10. We observed that only Vitamin E appeared to have a positive impact on the mitochondrial phosphorylation efficiency and on mitochondrial performance, namely on the ability to generate the electric transmembrane potential in the presence of supra-physiological calcium concentrations. Vitamin E administration also increased the mitochondrial concentration of Coenzyme Q10. None of the treatments was able to reverse the diabetic phenotype in GK rats. We conclude that in this model of mild hyperglycemia, administration of antioxidants may have a marginal positive impact on mitochondrial function.     

Ciprofibrate therapy in patients with hypertriglyceridemia and low high density lipoprotein (HDL)-cholesterol: greater reduction of non-HDL cholesterol in subjects with excess body weight (The CIPROAMLAT study)

Background

Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk.

Methods

The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion.

Results

In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% ± 11.32 vs. 39.74% ± 14.83 vs. 37.93% ± 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246).

Conclusion

The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.     

Meiotic inhibition with different cyclin-dependent kinase inhibitors in bovine oocytes and its effects on maturation and embryo development

Cyclin-dependent kinase inhibitors (CDKIs) such as butyrolactone I (BL-I) and roscovitine (ROS) maintain bovine oocytes blocked at the germinal vesicle (GV) stage. Bohemine (BOH), another CDKI, has been used for oocyte activation. The objective of this study was to determine whether BOH blocks meiosis and to compare its efficiency with other CDKIs (ROS and BL-I). Oocytes were cultured for 24 h in 0, 50, 100 and 150 microM BOH to determine the best concentration for blocking meiosis (experiment 1). GV rates were 3.3%, 64.5%, 83.3% and 88.9% (0,50, 100 and 150 microM, respectively). Experiment 2 compared meiotic inhibition efficiency of BOH (100 microM), ROS (25 microM) and BL-I (100 microM). BL-I presented the highest GV rates (97.5%). BOH and ROS were similar to each other (85.4% and 79.9%, respectively). To assess the reversibility of meiotic inhibition (experiment 3), oocytes underwent in vitro maturation (IVM) for 18 h after the 24 h inhibition. Control oocytes were submitted to IVM for 18 h (C18) or 24 h (C24). Maturation rates were either similar to (ROS and BL-I: 96.0% and 93.6%, respectively) or superior to (BOH, 96.9%) C24 (91.0%). All groups were superior to C18 (82.5%). In experiment 4, oocytes were treated as in experiment 3 and then in vitro fertilized and cultured for 8 days. Blastocyst rates for BL-I (32.3%) were similar to C24 (35.0%), while those for BOH (20.2%) and ROS (24.2%) were inferior. All groups were inferior to C18 (43.4%). The results show that: (a) BOH inhibits meiosis resumption; (b) BL-I is the most effective of the CDKIs tested for blocking meiosis; (c) culture of oocytes with meiosis inhibitors is fully reversible in terms of nuclear maturation but they may either decrease (BOH and ROS) or maintain (BL-I) embryo development rates.     

Overexpression of a synthetic gene encoding human alpha interferon in Escherichia coli

Interferons (IFNs) represent an important defense mechanism in vertebrates. In this work, we describe gene synthesis and assembly using the polymerase chain reaction as a method for single-step synthesis of DNA sequences. The oligonucleotides designed were based on Escherichia coli codon usage and two genes of IFN were synthesized: one containing a DNA sequence already known and the other, a mutated form in which two cysteine amino acid residues were replaced by serines in an attempt to improve the stability of the protein. DNA sequences were cloned into pAE, an E. coli vector that allows heterologous protein expression with or without a histidine tag. Recombinant human interferons (rhIFNs) were identified by Western blotting and ELISA using anti-human interferon polyclonal antibodies. Purification of the recombinant His-tagged proteins was achieved in a single step by Ni(2+)-charged column chromatography while proteins without His-tag were purified by extensively washing the inclusion bodies, the final yields being approximately 210 and 75mg/L, respectively. The rhIFNs expressed within this system were biologically active ( approximately 1,1x10(8)IU/mg) based on antiviral assay. The combined methodologies described here proved to be cost-effective and could be extended to other genes/proteins of interest.