In mouse macrophages (RAW 264.7 cells), toll-like receptor 4 (Tlr4) is a limiting factor in lipopolysaccharide (LPS) signal transduction. The expression of only 1-2 x 10(4) copies of recombinant Tlr4 per cell enhances sensitivity to LPS, shifting the EC50 by 30-fold to the left. Expression of the Tlr4(Lps-d) isoform of Tlr4 (found in C3H/HeJ mice) shifts the EC50 2600-fold to the right, essentially abolishing LPS responses. A truncated form of Tlr4, lacking a cytoplasmic domain, exerts only a weak inhibitory effect on signal transduction. Similarly, the normal or Tlr4(Lps-d) forms of protein lacking an ectodomain [corrected], cause modest inhibition of LPS signaling. Manipulations of Tlr4 structure and expression cause changes in LPS sensitivity that range over 3 to 4 orders of magnitude. These findings support the view that Tlr4 is an integral component of a solitary pathway for LPS signal transduction in macrophages and permit inferences related to the mechanism of signaling and its blockade.

Animals↗

PubMedDate not supplied

Neuropeptide Y (NPY), an endogenous presynaptic modulator of adrenergic neurotransmission in the rat vas deferens: structural and functional studies.

The role of neuropeptide tyrosine (NPY) on adrenergic neurotransmission was assessed in the rat vas deferens transmurally stimulated with square pulses of 0.15 or 15 Hz. Nanomoles of NPY inhibited the electrically-induced contractions on the prostatic half but not on the epididymal end of the ductus. NPY was at least 200-fold more potent than norepinephrine or adenosine to produce an equivalent inhibition. Complete amino acid sequence of NPY is required for full agonist activity; deletion of tyrosine at the amino terminus, i.e., NPY fragment 2-36 was 3-fold less potent than the native peptide. NPY fragment 5-36, 11-36 or 25-36 were proportionally less potent than NPY. Avian pancreatic polypeptide was inactive. The presynaptic nature of the NPY activity was established measuring the outflow of 3H-norepinephrine from the adrenergic varicosities of the vas deferens electrically stimulated. In this assay, NPY was more potent than NPY 2-36 or NPY fragment 5-36. No inhibitory action of NPY was detected in K+ depolarized tissues. The inhibitory effect of NPY on the rat vas deferens neurotransmission was not significantly modified by yohimbine, theophylline or naloxone, indicating that the effect of NPY is not due to the activation of alpha 2-adrenoceptors, adenosine receptors or opiate receptors respectively. Picrotoxin or apamin did not modify the inhibitory potency of NPY; verapamil or methoxyverapamil significantly reduced its potency. The inhibitory action of NPY is best explained through the activation of presynaptic NPY receptors that regulate norepinephrine release via a negative feedback mechanism. Structure activity studies give support to the notion of NPY receptors.

Animals↗

PubMedDate not supplied

A selective and sensitive kinetic method for the determination of procaine and benzocaine in pharmaceuticals.

A simple stopped-flow method for the determination of procaine and benzocaine which is suitable for their routine analysis in pharmaceutical samples is reported. The method is based on a condensation reaction between each compound and 4-dimethylaminocinnamaldehyde, which yields coloured compounds and which are monitored spectrophotometrically. The calibration graph generated is linear over the range 0.5-20 micrograms ml-1 (RSD 0.73%) for procaine and 0.5-15 micrograms ml-1 (RSD 0.40%) for benzocaine, and the selectivity is very high in both cases. The proposed methods were satisfactorily applied to the determination of the two drugs in various pharmaceutical samples. A procedure for resolution of procaine-benzocaine mixtures in mass ratios between 15:1 and 1:7 with a precision of ca 1% was developed in order to determine one compound in the presence of the other with no interference.

Benzocaine↗

PubMedDate not supplied

Safety and efficacy of a new flexible video laparoscope in diagnostic laparoscopy.

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Humans↗

PubMedDate not supplied

Purification, biochemical characterisation and partial primary structure of a new alpha-amylase inhibitor from Secale cereale (rye).

Plant alpha-amylase inhibitors show great potential as tools to engineer resistance of crop plants against pests. Their possible use is, however, complicated by the observed variations in specificity of enzyme inhibition, even within closely related families of inhibitors. Better understanding of this specificity depends on modelling studies based on ample structural and biochemical information. A new member of the alpha-amylase inhibitor family of cereal endosperm has been purified from rye using two ionic exchange chromatography steps. It has been characterised by mass spectrometry, inhibition assays and N-terminal protein sequencing. The results show that the inhibitor has a monomer molecular mass of 13,756 Da, is capable of dimerisation and is probably glycosylated. The inhibitor has high homology with the bifunctional alpha-amylase/trypsin inhibitors from barley and wheat, but much poorer homology with other known inhibitors from rye. Despite the homology with bifunctional inhibitors, this inhibitor does not show activity against mammalian or insect trypsin, although activity against porcine pancreatic, human salivary, Acanthoscelides obtectus and Zabrotes subfasciatus alpha-amylases was observed. The inhibitor is more effective against insect alpha-amylases than against mammalian enzymes. It is concluded that rye contains a homologue of the bifunctional alpha-amylase/trypsin inhibitor family without activity against trypsins. The necessity of exercising caution in assigning function based on sequence comparison is emphasised.

Animals↗

PubMedDate not supplied

Use of pruned computational neural networks for processing the response of oscillating chemical reactions with a view to analyzing nonlinear multicomponent mixtures.

The suitability of pruned computational neural networks (CNNs) for resolving nonlinear multicomponent systems involving synergistic effects by use of oscillating chemical reaction-based methods implemented using the analyte pulse perturbation technique is demonstrated. The CNN input data used for this purpose are estimates provided by the Levenberg-Marquardt method in the form of a three-parameter Gaussian curve associated with the singular profile obtained when the oscillating system is perturbed by an analyte mixture. The performance of the proposed method was assessed by applying it to the resolution of mixtures of pyrogallol and gallic acid based on their perturbating effect on a classical oscillating chemical system, viz. the Belousov-Zhabotinskyi reaction. A straightforward network topology (3:3:2, with 18 connections after pruning) allowed the resolution of mixtures of the two analytes in concentration ratios from 1:7 to 6:2 with a standard error of prediction for the testing set of 4.01 and 8.98% for pyrogallol and gallic acid, respectively. The reduced dimensions of the selected CNN architecture allowed a mathematical transformation of the input vector into the output one that can be easily implemented via software. Finally, the suitability of response surface analysis as an alternative to CNNs was also tested. The results were poor (relative errors were high), which confirms that properly selected pruned CNNs are effective tools for solving the analytical problem addressed in this work.

Journal Article↗