New insights for using self-assembly materials to improve the detection stability in label-free DNA-chip and immuno-sensors

This paper examines reliable advancements in low-cost DNA- and immuno-chips. Capacitance detection was successfully chosen to develop label-free bio-chips. Probe immobilization was rigorously investigated in order to obtain reliable capacitance measurements. Protein probes immobilized by using usual alkanethiols or thiolated ssDNA probes directly immobilized on gold do not allow sufficient stable capacitance measurements. New alkanethiols improved with ethylene–glycol function are shown in this paper to be more suitable materials for capacitive bio-chip development. Atomic Force Microscopy, Quartz Crystal Microbalance, and Capacitance Measurements were used to demonstrate that ethylene–glycol alkanethiols allow high time stability, smaller errors in detection, and improved ideal behaviour of the sensing surfaces. Measured capacitance is in the range of 8–11 nF/mm² for antibody layers and close to 6 nF/mm² for DNA probes. It is in the range of 10–12 nF/mm² and of 4–6 nF/mm² for antigen and DNA detection, respectively. The percentage error in detection is highly improved and it is in the range of 11–37% and of 0,23–0,82% for antigen and DNA, respectively. The reproducibility is also improved and it is close to 0,44% for single spot measurements on ethylene–glycol alkanethiols. A molecular theory attributing these improvements to water molecules strongly coordinated by ethylene–glycol functional groups and to solution ions not entering into probe films is finally proposed.     

Regulation of RANKL by biomechanical loading in fibrochondrocytes of meniscus

OBJECTIVE: We sought to determine whether fibrochondrocytes from menisci express receptor activator of NF-kappaB (RANK), its ligand (RANKL), or osteoprotegerin (OPG) and, if so, whether their expression is modulated by dynamic mechanical loading under inflammatory and normal conditions. METHODS: Fibrochondrocytes from rat menisci were subjected to cyclic tensile strain (CTS) at various magnitudes and frequencies in the presence or absence of interleukin (IL)-1beta for up to 24 h. In order to determine whether a possible regulatory effect of mechanical loading on RANKL and its receptors under inflamed conditions is sustained, cells were stimulated with IL-1beta for 24 h while being subjected to CTS only for the initial 4 and 8h, respectively. Regulation of RANKL, RANK, and OPG expression and synthesis were determined by semiquantitative and real-time PCR, Western blotting, and immunofluorescence. RESULT: Fibrochondrocytes constitutively expressed low levels of RANKL and RANK but marked levels of OPG. IL-1beta upregulated expression and synthesis of RANKL and RANK significantly (p<0.05), whereas expression of OPG was unaffected following 4 and 24 h. When fibrochondrocytes were simultaneously subjected to CTS and IL-1beta, expression of RANKL and RANK was significantly (p<0.05) downregulated as compared to that of IL-1beta-stimulated unstretched cells. The inhibitory effect of CTS on the IL-1beta-induced upregulation of RANKL and RANK was sustained as well as magnitude and frequency dependent. CONCLUSIONS: Our study provides evidence that RANKL and its receptors are expressed in fibrochondrocytes from meniscus. These data also demonstrate that dynamic mechanical loading can modify the expression of RANKL and RANK in inflammatory conditions.     

Protein deiminases: new players in the developmentally regulated loss of neural regenerative ability

Spinal cord regenerative ability is lost with development, but the mechanisms underlying this loss are still poorly understood. In chick embryos, effective regeneration does not occur after E13, when spinal cord injury induces extensive apoptotic response and tissue damage. As initial experiments showed that treatment with a calcium chelator after spinal cord injury reduced apoptosis and cavitation, we hypothesized that developmentally regulated mediators of calcium-dependent processes in secondary injury response may contribute to loss of regenerative ability. To this purpose we screened for such changes in chick spinal cords at stages of development permissive (E11) and non-permissive (E15) for regeneration. Among the developmentally regulated calcium-dependent proteins identified was PAD3, a member of the peptidylarginine deiminase (PAD) enzyme family that converts protein arginine residues to citrulline, a process known as deimination or citrullination. This post-translational modification has not been previously associated with response to injury. Following injury, PAD3 up-regulation was greater in spinal cords injured at E15 than at E11. Consistent with these differences in gene expression, deimination was more extensive at the non-regenerating stage, E15, both in the gray and white matter. As deimination paralleled the extent of apoptosis, we investigated the effect of blocking PAD activity on cell death and deiminated-histone 3, one of the PAD targets we identified by mass-spectrometry analysis of spinal cord deiminated proteins. Treatment with the PAD inhibitor, Cl-amidine, reduced the abundance of deiminated-histone 3, consistent with inhibition of PAD activity, and significantly reduced apoptosis and tissue loss following injury at E15. Altogether, our findings identify PADs and deimination as developmentally regulated modulators of secondary injury response, and suggest that PADs might be valuable therapeutic targets for spinal cord injury.     

Relative acidity scale of glycine- and taurine-conjugated bile acids through ESI-MS measurements

The most important bile acids, in the form of glycine and taurine conjugates, have been ordered in terms of relative acidity scale. The measurements have been carried out using mass spectrometric techniques. The group of taurine conjugates confirm the superior acidity over the glycine derivatives. Rationale of the differences found in gas-phase and comparison with the data reported in solution-phase are discussed with the support of theoretical calculations. The study has been completed with the acidity sequence of mixed oxo-hydroxy bile acids.     

Compensatory expression and substrate inducibility of gamma-glutamyl transferase GGT2 isoform in Arabidopsis thaliana

γ-Glutamyl transferases (GGT; EC 2.3.2.2) are glutathione-degrading enzymes that are represented in Arabidopsis thaliana by a small gene family of four members. Two isoforms, GGT1 and GGT2, are apoplastic, sharing broad similarities in their amino acid sequences, but they are differently expressed in the tissues: GGT1 is expressed in roots, leaves, and siliques, while GGT2 was thought to be expressed only in siliques. It is demonstrated here that GGT2 is also expressed in wild-type roots, albeit in very small amounts. GGT2 expression is enhanced in ggt1 knockout mutants, suggesting a compensatory effect to restore GGT activity in the root apoplast. Supplementation with 100 μM glutathione (GSH) resulted in the up-regulation of GGT2 gene expression in wild-type and ggt1 knockout roots, and of GGT1 gene expression in wild-type roots. Glutathione recovery was hampered by the GGT inhibitor serine/borate, suggesting a major role for apoplastic GGTs in this process. These findings can explain the ability of ggt1 knockout mutants to retrieve exogenously added glutathione from the growth medium.     

Metabolic alterations in K562 cells exposed to taxol and tyrphostin AG957: 1H NMR and biochemical studies

K562 cells exposed for 3 h to taxol or taxol plus tyrphostin AG957 exhibited a significant variation in the concentration of the water-soluble metabolites glutathione, myo-inositol and phosphorylcholine, as evaluated by (1)H NMR up to 72 h incubation in drug-free medium. Cells treated with both drugs showed an increase of glutathione and glutathione reductase at 24 h and a sharp decrease of myo-inositol between 8 and 24 h. Phosphorylcholine increased at 8 h both in taxol and taxol plus AG957-treated cells, which was then abruptly inverted to a significantly lower concentration at 24 h, subsequently increasing again to values higher than those found in taxol-treated and control cells. All the above reported effects were lacking in cells exposed to AG957 alone. These modifications, despite the enhancement of the overall apoptotic cascade in taxol plus AG957-treated cells, can be related to the activation of cellular detoxification mechanisms, to the correct osmolarity maintenance, and to alterations of phospholipid metabolism.     

Determination of ethyl-p-hydroxybenzoate in sow pancreatic juice by reversed-phase high-performance liquid chromatography

We have developed a high-performance liquid chromatographic–UV–Vis–diode-array detection (HPLC–DAD) method for the determination of ethyl-p-hydroxybenzoate, a hydrolytic degradation product of the synthetic protease inhibitor, gabexate-mesilate ethyl-p-(6-guanidinohexanoyloxy) benzoate methanesulfonate (GM) (FOY) in sow pancreatic juice. Methyl-p-hydroxybenzoate (I) was used as the internal standard. The pancreatic juice was deproteinised by acetonitrile and the analytes were chromatographed on a reversed-phase C₁₈ LC column using the gradient elution method. The mobile phase consisted of a solution of 0.017 M orthophosphoric acid and another solution of acetonitrile–water (80:20, v/v). The wavelength of detection was 237 nm. The limit of quantification of the method was 0.20 μM at a 9:1 signal-to-noise ratio. The overall intra- and inter-day accuracy (relative error, RE) ranged from 14.2 to 8.3% and from 13.3 to 9.8, respectively. The overall intra- and inter-day precision (relative standard deviation, RSD) ranged from 7.6 to 2.62% and from 6.7 to 3.1%, respectively. The method proved to be sensitive, specific, accurate and precise and was successfully used to determine the ethyl-p-hydroxybenzoate (II) in sow pancreatic juice.     

Biochemical and genetic analysis of Streptococcus mutans alpha-galactosidase.

The aga gene coding for alpha-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage lambda. The gene was subcloned into plasmid vectors and shown to specify a novel protein of Mr 80,000. Characterization of alpha-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the alpha-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted Mr of 82,022 and revealed regions of homology to alpha-galactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all alpha-galactosidase activity and abolished the ability to ferment melibiose; alpha-glucosidase activity was also lost, due to an indirect effect on the dexB gene.     

Growth factor receptors gene expression and Akt phosphorylation in benign human thyroid nodules are unaffected by chronic thyrotropin suppression

Levothyroxine (L-T4)-based suppression of thyrotropin (TSH) secretion is widely used to prevent the growth of benign thyroid nodules, although the effectiveness of this approach has been demonstrated only in a subset of patients. In this study, we analyzed the in vivo effects of L-T4-mediated TSH suppression on elements of insulin/IGF-1-dependent growth-regulating pathways in tissues from patients with benign thyroid nodules. Nodular and non-nodular tissue specimens were collected from 63 patients undergoing thyroidectomy. 32 had received preoperative TSH suppressive therapy with TSH levels consistently below 0.5 mU/l (L-T4 group). TSH suppression had not been used in the other 31, and their TSH levels were normal (0.8-4 mU/l (control group). Quantitative RT-PCR was used to measure mRNA levels for TSH receptor, IGF1, IGF-1 receptor, insulin receptor, insulin receptor substrate 1 in nodular and non-nodular tissues from the 2 groups. Akt and phosphorylated Akt protein levels were detected by Western blot. Mean levels of mRNA for all genes tested were similar in the 2 groups, in both nodular and non-nodular tissues. The 2 groups were also similar in terms of phosphorylated Akt protein levels (measured by densitometric scan in 10 randomly selected nodules from each group). This is the first demonstration based on the study of human thyroid tissues that TSH suppression does not affect the expression of components of the insulin/IGF-1-dependent signaling pathways regulating thyrocyte growth. This may explain the lack of effectiveness of TSH-suppressive therapy in a substantial percentage of benign thyroid nodules.