Extrinsic control of the release of galanin and VIP from intrinsic nerves of isolated, perfused, porcine ileum.

By immunohistochemistry galanin-like immunoreactivity and vasoactive intestinal polypeptide (VIP)-like immunoreactivity were found in nerve cell bodies mostly in the submucous plexus and in nerve fibres in the mucosa, submucosa and muscularis including the myenteric plexus of the porcine ileum and were found to co-exist in most of these structures. Using isolated, perfused porcine ileum we studied the release of galanin and VIP in response to electrical stimulation of the mixed periarterial nerves or to intraarterial infusions of different neuroactive agents. Nerve stimulation (4-10 Hz) inhibited the basal release of galanin and VIP from the ileum (to 69 +/- 6 and 62 +/- 6% of basal release). After infusion of the alpha-adrenergic blocker, phentolamine, (10(-6) M) electrical stimulation increased the release of both galanin and VIP (to 140 +/- 12 and 133 +/- 13% of basal output). This increase was abolished by atropine (10(-6) M) and by hexamethonium (3.10(-5) M). Infusion of norepinephrine (10(-6) M) inhibited, whereas acetylcholine (10(-6) M) stimulated the release of both peptides. The effect of the latter was abolished by atropine. The inhibitory effect of nerve stimulation was not influenced by atropine. Our results suggest that the galanin- and VIP-producing intrinsic neurons receive inhibitory signals by noradrenergic nerve fibers and stimulatory signals mediated by cholinergic nerves, possibly via a cholinergic interneuron.     

Randomly amplified polymorphic DNA (RAPD) profiling of Plasmodiophora brassicae

M. MÖLLER AND R. HARLING. 1996. A technique is described for the preparation of DNA suitable for use in RAPD analysis from pure, sterile resting spores of Plasmodiophora brassicae . Using this technique, random 10-base pair primers were applied to P. brassicae DNA from three single spore isolates. The resulting profiles were compared with the race classification based on inoculation of the European Clubroot Differential (ECD) series of Brassica hosts. Out of 40 primers tested, 23 gave amplification products, three gave isolate-specific profiles and one a profile which corresponded with the ECD race classification of the isolates. RAPD profiling can provide a faster means of race classification in P. brassicae .     

Protein-Lipopolysaccharide Complexes Produced by Virulent and Non-Virulent Isolates of Verticillium albo-atrum and V. dahliae are Non-Specific Toxins to Tomato and Potato

Toxic protein-lipopolysaccharide complexes (PLPC) were isolated from culture filtrates of Verticillium albo-atrum (isolate WCS 800) and V. dahliae (WCS 070) isolates, both virulent to tomato and potato cultivars, and from an isolate of,V. albo-atrum (V22W) which was non-virulent to these hosts. The virulent isolates each produced one major PLPC in culture and the non-virulent isolate produced two (fractions 1 and 2, characterized by their elution pattern after gel filtration). Virulence in these three isolates was not related to quantity of PLPC produced in culture. However, PLPC from the virulent isolates were toxic to tomato in a leaf bioassay at 4μg ml^-1 (WCS 800) and 20μg ml^-1 (WCS 070) but the two PL, PC from the non-virulent isolate required concentrations of 100 and 1000 μg ml^-1 for toxicity. Production of a modified, less toxic PLPC in V22W may partly account for its non-virulence. Gel filtration of PLPC from the three isolates on a calibrated Sephacryl S-400 column, eluted in phosphate buffer plus NaCl, indicated a compound of heterogeneous molecular mass with an average of 126,000 daltons for the PLPC of WCS 800, WCS 070 and, fraction 1 of V22W, and 25,000 daltons for fraction 2 of V22W. Attempts to extract a low molecular weight toxin from the PLPC by extended dialysis were unsuccessful. The susceptibility of 12 tomato and 19 potato cultivars to the Verticillium isolates was compared with their sensitivity to PLPC. Susceptibility was not correlated with toxin sensitivity and the PLPC were concluded to be non-specific toxins in these hosts.     

Naturally occurring products of proglucagon 111-160 in the porcine and human small intestine

Recent studies have revealed that the glucagon gene is expressed in the mammalian intestine. Here it codes for "glicentin" (proglucagon 1-69) and a glucagon-like peptide, proglucagon 78-107, recently isolated from porcine intestine. We studied the fate of the remaining COOH-terminal part of proglucagon (proglucagon 111-160) using radioimmunoassays against proglucagon 111-123 and 126-160. Two peptides were isolated from acid ethanol extracts of porcine ileal mucosa and sequenced: one corresponding to proglucagon 126-158 and one probably corresponding to proglucagon 111-158. By comparing human and porcine proglucagon sequences, Ala117 is replaced by Thr, and Ile138, Ala144, Ile152 and Gln153 are replaced by Val, Thr, Leu, and His. By gel filtration and radioimmunoassay of intestinal extracts it was established that a large part of porcine and virtually all of human proglucagon are processed to release proglucagon 111-123 (designated spacer peptide 2), which, like proglucagon 126-158 must be considered a potential hormonal entity. By isocratic high pressure liquid chromatography human spacer peptide 2 was indistinguishable from synthetic proglucagon 111-122 amide, suggesting that this is the structure of the naturally occurring human peptide.     

Conditions for Efficient Isolation and Regeneration of Protoplasts from Fulvia fulva

A procedure for producing high yields and high regeneration frequencies of protoplasts from Fulvia fulva is described. This procedure was devised by systematic trials of various parameters, such as culture age and osmotic support, which are known to affect protoplast yield and regeneration. Mycelium from liquid cultures of 24–48 h, incubated with Novozym 234 in buffered 1.0 M MgSO₄, gave the best conditions for protoplast release. Regeneration frequencies up to 50% were obtained with a complete medium containing 0.8 M sucrose for osmotic support.     

Discovery of Novel Irreversible Inhibitors of Interleukin (IL)-2-inducible Tyrosine Kinase (Itk) by Targeting Cysteine 442 in the ATP Pocket

IL-2-inducible tyrosine kinase (Itk) plays a key role in antigen receptor signaling in T cells and is considered an important target for anti-inflammatory drug discovery. In order to generate inhibitors with the necessary potency and selectivity, a compound that targeted cysteine 442 in the ATP binding pocket and with an envisaged irreversible mode of action was designed. We incorporated a high degree of molecular recognition and specific design features making the compound suitable for inhaled delivery. This study confirms the irreversible covalent binding of the inhibitor to the kinase by x-ray crystallography and enzymology while demonstrating potency, selectivity, and prolonged duration of action in in vitro biological assays. The biosynthetic turnover of the kinase was also examined as a critical factor when designing irreversible inhibitors for extended duration of action. The exemplified Itk inhibitor demonstrated inhibition of both T_H1 and T_H2 cytokines, was additive with fluticasone propionate, and inhibited cytokine release from human lung fragments. Finally, we describe an in vivo pharmacodynamic assay that allows rapid preclinical development without animal efficacy models.     

PCR-based detection of the polymorphism at codon 136 in the ovine prion protein gene

In many breeds of sheep, a polymorphism at codon 136 of the prion protein gene has been shown to be strongly associated with the risk of developing scrapie. A single-step procedure for detecting this allelic variation is described here. When performed on a series of animals, the test was in complete agreement with their genotypes as had been previously determined by sequencing. The test is potentially easier and quicker to perform than any of the variety of methods that are currently used for this purpose.