A portable microelectrode array recording system incorporating cultured neuronal networks for neurotoxin detection

Cultured neuronal networks, which have the capacity to respond to a wide range of neuroactive compounds, have been suggested to be useful for both screening known analytes and unknown compounds for acute neuropharmacologic effects. Extracellular recording from cultured neuronal networks provides a means for extracting physiologically relevant activity, i.e. action potential firing, in a noninvasive manner conducive for long-term measurements. Previous work from our laboratory described prototype portable systems capable of high signal-to-noise extracellular recordings from cardiac myocytes. The present work describes a portable system tailored to monitoring neuronal extracellular potentials that readily incorporates standardized microelectrode arrays developed by and in use at the University of North Texas. This system utilizes low noise amplifier and filter boards, a two-stage thermal control system with integrated fluidics and a graphical user interface for data acquisition and control implemented on a personal computer. Wherever possible, off-the-shelf components have been utilized for system design and fabrication. During use with cultured neuronal networks, the system typically exhibits input referred noise levels of only 4-6 microVRMS, such that extracellular potentials exceeding 40 microV can be readily resolved. A flow rate of up to 1 ml/min was achieved while the cell recording chamber temperature was maintained within a range of 36-37 degrees C. To demonstrate the capability of this system to resolve small extracellular potentials, pharmacological experiments with cultured neuronal networks have been performed using ion channel blockers, tetrodotoxin and tityustoxin. The implications of the experiments for neurotoxin detection are discussed.     

Electrical frequency dependent characterization of DNA hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of electrochemical impedance spectroscopy (EIS) and quartz crystal microgravimetry (QCM). Synthetic 5'-amino terminated single stranded oligonucleotides (ssDNA) were attached to the exposed glass surface between the digits of microlithographically fabricated interdigitated microsensor electrodes using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA immobilization was achieved to the surface of the gold driving electrodes of AT-cut quartz QCM crystals using 3-mercaptopropyl-trimethoxysilane. Significant changes in electrochemical impedance values (both real and imaginary components) (11% increase in impedance modulus at 120 Hz) and resonant frequency values (0.004% decrease) were detected as a consequence of hybridization of the bound ssDNA upon exposure to its complement under hybridization conditions. Non-complementary (random) sequence sowed a modest decrease in impedance and a non-detectable change in resonant frequency. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA, has been demonstrated. The potential for development of label-free, low density DNA microarrays is demonstrated and is being pursued.     

The Arabidopsis TDS4 gene encodes leucoanthocyanidin dioxygenase (LDOX) and is essential for proanthocyanidin synthesis and vacuole development

The anthocyanin and proanthocyanidin (PA) biosynthetic pathways share common intermediates until leucocyanidin, which may be used by leucoanthocyanidin dioxygenase (LDOX) to produce anthocyanin, or the enzyme leucoanthocyanidin reductase (LAR) to produce catechin, a precursor of PA. The Arabidopsis mutant tannin deficient seed 4 (tds4-1) has a reduced PA level and altered pattern PA accumulation. We identified the TDS4 gene as LDOX by complementation of the tds4-1 mutation either with a cosmid encoding LDOX or a 35S:LDOX construct. Independent Arabidopsis lines with a T-DNA insertion in the LDOX gene had a similar phenotype, and one was allelic to tds4-1. The seed phenotype of ban tds4 double mutants showed that LDOX precedes BANYULS (BAN) in the PA pathway, confirming recent biochemical characterisation of BAN as an anthocyanidin reductase. Double mutant analysis was also used to order the other TDS genes. Analysis of the PA intermediates in tds4-1 revealed three dimethylaminocinnamaldehyde (DMACA) reacting compounds that accumulated in extracts from developing seeds. Analysis of Arabidopsis PA and its precursors indicates that Arabidopsis, unlike many other plants, exclusively uses the epicatechin and not the catechin pathway to PA. Transmission electron microscopy (TEM) showed that the pattern observed when seeds of tds4 were stained with DMACA was a result of the accumulation of PA intermediates in the cytoplasm of endothelial cells. Fluorescent marker dyes were used to show that tds4 endothelial cells had multiple small vacuoles, instead of a large central vacuole as observed in the wild types (WT). These results show that in addition to its established role in the formation of anthocyanin, LDOX is also part of the PA biosynthesis pathway.     

4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle

Maintenance of cellular protein stores in skeletal muscle depends on a tightly regulated synthesis-degradation equilibrium that is conditionally modulated under an extensive range of physiological and pathophysiological circumstances. Recent studies have established the initiation phase of mRNA translation as a pivotal site of regulation for global rates of protein synthesis, as well as a site through which the synthesis of specific proteins is controlled. The protein synthetic pathway is exquisitely sensitive to the availability of hormones and nutrients and employs a comprehensive integrative strategy to interpret the information provided by hormonal and nutritional cues. The translational repressor, eukaryotic initiation factor 4E binding protein 1 (4E-BP1), and the 70-kDa ribosomal protein S6 kinase (S6K1) have emerged as important components of this strategy, and together they coordinate the behavior of both eukaryotic initiation factors and the ribosome. This review discusses the role of 4E-BP1 and S6K1 in translational control and outlines the mechanisms through which hormones and nutrients effect changes in mRNA translation through the influence of these translational effectors.     

Modelling Metal Complexes of Calixarene Esters and Phosphine Oxides Using Molecular Mechanics

This paper focuses on the molecular modelling of a number of calixarene ester and phosphine oxide metal ion complexes. Monte Carlo conformational searches, in conjunction with the Merck Molecular Force Field, were carried out using Spartan SGI Version 5.0.1. running on Silicon Graphics O2 workstations. In the case of the calix[4]arene tetraesters, the optimised models strongly suggest that the selectivity of these ligands is strongly related to the eight-fold nature of the coordination with the Na⁺ ion, while coordination with the Li⁺ ion, for example, is merely three-fold. This feature of eight-fold coordination is also observed in the models of the complexes formed by the calix[4]arene tetraphosphine oxides with calcium. However, whereas the eight-fold coordination is unique to the model of the TPOL:Ca²⁺ complex among the ions modelled, this mode of coordination occurs for TPOS with sodium and potassium, in addition to calcium. This concurs with the observation that calcium selectivity is obtained with ion selective electrodes based on TPOL but not TPOS. Though the cavity in the calix[5]arenes PPOL and PPOLx and the calix[6]arene HPOL, in their uncomplexed form, are much larger than that of the corresponding calix[4]arenes, the pattern of selectivity is the same – the ligands are selective for calcium. The models of the complexes of these larger calixarenes, such as PPOL:Ca²⁺, strongly suggest that the reason for this similarity is that four of the available phosphine oxide groups complex with the calcium ion, and the others are forced away from the cavity region for steric reasons. The resulting eight-fold coordination, is therefore, similar to that of the calix[4]arenes studied.Electronic Supplementary Material available.     

Apolipoprotein-E dependent role for the FAS receptor in early onset Alzheimer's disease: finding of a positive association for a polymorphism in the TNFRSF6 gene

The TNFRSF6 gene encodes FAS, a cell-surface receptor involved in apoptosis initiation. Elevated levels of FAS have been reported in the brains of Alzheimer's disease (AD) patients. We have tested a G/A polymorphism at position -670 in the TNFRSF6 gene for association with non-familial, early onset Alzheimer's disease (EOAD) by using dynamic allele-specific hybridization. In an initial set of Scottish EOAD cases (n=78) and controls (n=152), we found that, for individuals carrying one or two APOE4 alleles, the homozygous GG-genotype was enriched in the patients (26.7% versus 10.9% in controls). A second study was conducted on an independent set of Scottish individuals (87 EOAD, 358 controls). In this material, the TNFRSF6 GG-genotype frequency was elevated in patients regardless of APOE4 status (28.7% versus 15.1%) and was even more enriched in APOE4 carriers (35.9% versus 15.3%). A combination of the two sample sets (165 cases, 510 controls) gave a significant disease association for the TNFRSF6 GG-genotype that was irrespective of APOE4 (P=0.0020) and that was almost completely attributable to the enrichment present within the set of APOE4 carriers (P=0.0016). This represents an odds ratio of 8.71 for GG-homozygotes carrying at least one APOE4 allele compared with other TNFRSF6 genotypes in APOE4 non-carriers. The TNFRSF6 variation was further explored in Scottish late-onset Alzheimer's disease (n=159) but no associations were found. These results imply that TNFRSF6, in interaction with APOE4, is a genetic risk factor for sporadic EOAD. Hence, the AD risk contributed by APOE4 could be mechanistically related to a pathway in common with FAS-mediated apoptosis.     

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.     

Molecular changes in the d-bifunctional protein cDNA sequence in australasian patients belonging to the bifunctional protein complementation group

The cDNA sequence for the human d-bifunctional protein (D-BP: 17β-hydroxysteroid dehydrogenase IV) was investigated in patients with peroxisomal disorders belonging to the BP complementation group (CG). In three cases, analysis of polymerase chain reaction products generated from the patients' cDNA indicated the presence of a deletion within the region corresponding to nucleotides 209–537 of the normal cDNA sequence. Subsequent sequencing revealed that, in two of the patients, 47 base pairs were missing, with the deletion corresponding to nucleotides 302/3–349/50 of the normal sequence. In the third patient, a smaller deletion of 22 bp (nucleotides 280/1–302/3) was characterized. Only the mutant sequence was detected in each of these cases, consistent with parental consanguinity. Both deletions cause a frameshift, and would lead to premature termination of the BP. Available family members were also investigated, and the findings conformed with expectations for an autosomal recessive disorder. In addition to the deletions, a number of other base changes have been identified in this series of patients. In particular, one patient, whose parents were also consanguineous, was homozygous for a base change, which results in a nonconservative substitution of serine 177 with a phenylalanine residue. The functional significance of this amino acid substitution, as well as the other identified changes, is still to be determined. Nevertheless, our data provide strong support for the hypothesis that defects in the gene for the D-BP are responsible for the β-oxidation defect in patients belonging to the BP CG.