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Mastitis in cattle is an inflammation of the udder that can be caused by a number of bacterial pathogens. Standard testing requires a series of tests to accurately identify the cause. Mastitic milk cannot be sold and the treatment usually involves a course of antibiotics during which time the milk can also not be sold so a fast identification of the pathogen involved is essential to reduce lost revenue. A DNA microarray was designed to identify the pathogens that could be responsible for mastitis. A 6 x 6 grid of oligonucleotides was covalently immobilised (by use of a surface attached polymer network) onto the untreated surface of each well of a 96 well microtitre plate [1] . DNA extracted from mastitic milk samples was amplified using Cy5 labelled primers and subsequently hybridised to the immobilised probes on the microarray. A standard ELISA plate washer was used to wash the microtitre plate and the signals from the bound PCR products were read in a commercially available reader (FLAIR). Using the species probes that give the strongest and weakest signals, S.Aure_35p (Staphylococcus aureus) and E.coli_448p (Escherichia coli) respectively the hybridisation signal variation from well to well was investigated The microarray can be regenerated (removal of the bound amplification products) by a hot wash cycle and then reused. To test the stability of the probes over subsequent cycles of hybridisations and regenerations a Cy3 labelled oligonucleotide was added to all the spots printed on the array and the array was subjected to 10 rounds of mock hybridisation and regeneration cycles. After 10 cycles there was only a minor loss of signal.

Stored endogenous acetylcholine (ACh) and in vitro synthesis of [3H]ACh were measured in isolated, mucosa-intact and mucosa-denuded airways of rat, guinea pig, and humans. In addition, choline acetyltransferase (ChAT) activity and ACh content were measured in freshly isolated airway mucosa as well as in cultured epithelial cells of rat tracheas. Rat tracheas stored 25 nmol/g ACh, whereas guinea pig tracheas and human bronchi contained only 2-3 nmol/g ACh. When incubated with [3H]choline, the isolated airways of rat, guinea pig, and human synthesized significant amounts of [3H]ACh. In guinea pig and human airways, removal of the mucosa affected neither stored ACh nor in vitro synthesis of [3H]ACh. In rat tracheas, however, removal of the mucosa resulted in a 50% reduction of stored ACh. Freshly isolated mucosa wiped off from the luminal surface of rat tracheas contained large amounts of ACh (6.5 nmol/g airway), whereas in human mucosa (central bronchi) only small amounts of ACh were found. In enzymatically isolated mucosal cells of rat tracheas, a considerable ChAT activity (21 nmol.mg protein 1.h-1) was detected, blockable by bromoacetylcholine. Enzymatically isolated human mucosa contained a rather low ChAT-like activity (0.5 nmol.mg protein 1.h-1), not sensitive to bromoacetylcholine. In cultured epithelial cells of rat tracheas (4th-6th passage), neither ChAT activity nor ACh was detected. The large airways of rat, guinea pig, and humans contain considerable amounts of ACh, supporting histological evidence of a dense cholinergic innervation, particularly of rat tracheas. The mucosa of rat tracheas synthesizes and stores large amounts of ACh, whereas the low ChAT activity in human mucosa argues against the presence of cholinergic neurons able to synthesize and store ACh.

Summary The variability of nine different isozyme systems, chromosome number, colour of fresh and dried latex and presence or absence of meconic acid was investigated in altogether 45 populations of Papaver dubium and P. lecoqii mainly from C Europe. It was found that P. dubium is hexaploid with 2n = 42 chromosomes, has brown dried latex, and contains meconic acid. P. lecoqii is tetraploid with 2n = 28 chromosomes, has red dried latex, and lacks meconic acid. The two taxa can also be distinguished by differences in three of the nine isozymes analysed, and are almost completely monomorphic for their respective isozyme pattern. It is concluded that the two taxa, which cannot be reliably distinguished with morphological characters, should be treated as species. The results are shortly discussed with reference to the ecology of the two taxa as colonizers of man-made habitats.

Receptor-mediated regulation of acetylcholine release in the airways, particularly in humans, remains unclear. In the present study, the tissue content of acetylcholine and release of [3H]acetylcholine were measured in freshly dissected human bronchi obtained at thoracotomy. Large (main and lobar bronchi) and small (segmental and subsegmental bronchi) airways contained considerable amounts of endogenous acetylcholine (300 +/- 50 pmol/100 mg wet weight), whereas significantly less was found in lung parenchyma (60 +/- 30 pmol/100 mg). Isolated small bronchi incubated in an organ bath with the precursor [3H]choline synthesized significant amounts of [3H]acetylcholine (26,000 +/- 4,000 dpm/100 mg). Subsequent transmural stimulation (four 20 s trains at 15 Hz) of radiolabeled bronchi caused an enhanced tritium outflow that was abolished by removal of extracellular calcium or by tetrodotoxin. HPLC analysis of the medium collected before, during, and after transmural stimulation showed that the electrically stimulated tritium outflow represented exclusively [3H]acetylcholine, whereas the outflow of [3H]choline and [3H]phosphorylcholine was not affected by electrical stimulation. Oxotremorine (0.1 and 1 mumol/L) inhibited evoked [3H]acetylcholine release in a concentration-related manner, whereas atropine (0.03 mumol/L) enhanced evoked [3H]acetylcholine release. Inactivation of cyclooxygenase activity by 3 mumol/L of indomethacin did not impair the inhibitory effect of 0.1 or 1 mumol/L of oxotremorine. In conclusion, the present experiments indicate a considerable cholinergic innervation of human large and small airways.

Microarray based test assays have become increasingly important tools in diagnostics for fast multi-parameter detection especially where sample volumes are limited. We present here a simple procedure to create polysaccharide microarrays, which can be used to analyze antibodies using an integrated, complementary metal-oxide-semiconductor (CMOS) based electric signal readout process. To accomplish this chips are used which consist of an array of silicon photodiodes and where different types of polysaccharides from the bacteria Streptococcus pneumoniae are printed on the (silicon dioxide) chip surface. Typical amounts of polysaccharide deposited in the printing process are around 12 attomol/spot. In a subsequent reaction step the polysaccharide microarrays were used for the measurement of IgG antibody concentrations in human blood sera using either chemiluminescence or fluorescence based detection. To understand the device performance the influence of surface density of the immobilized polysaccharide molecules and other parameters on the assay performance are investigated. The dynamic measurement range of the sensor is shown to reach over more than 3 decades of concentration and covers the whole physiologically relevant range for the analysis of antibodies against a large panel of pneumococcal polysaccharides.

Primary cultures of human bronchial epithelial cells (HBE-cells) were established to measure granulocyte-macrophage colony stimulating factor (GM-CSF) release. HBE-cells showed a basal GM-CSF release (82+/-20 ng/well/24 h; 30 donors), which was increased by interleukin-1 beta(IL-1beta, 1 ng/ml) by 270%. This effect was blocked by 1 microM dactinomycin or 10 microM cycloheximide, i.e. the stimulatory effect of IL-1beta depended on de-novo synthesis. Histamine (100 microM) and acetylcholine ( 100 nM) stimulated GM-CSF release more than two-fold above the baseline. Nicotine (1 microM) increased GM-CSF release to a similar extent, and this effect was prevented by 30 microM (+)-tubocurarine. The stimulatory effect was attenuated or even lost with high agonist concentrations (10 microM acetylcholine; 100 microM nicotine) suggesting receptor desensitization. The muscarinic receptor agonist oxotremorine did not affect GM-CSF release. Serotonin, substance P and calcitonin-gene related peptide had no effect on GM-CSF release. In conclusion, acetylcholine can trigger GM-CSF release from human airway epithelial cells via stimulation of nicotinic receptors.

In the present study we demonstrate that acetylcholine is synthesized by cultured mammalian glial cells identified by cell-type specific markers. Primary cultures of rat brain astrocytes or microglia contained 2.0 and 1.6 pmol acetylcholine/10(6) cells on average respectively. Astrocyte cultures established from neonatal mouse brain contained even more acetylcholine (about 80 pmol acetylcholine/10(6) cells). Primary cultures of rat brain astrocytes showed choline acetyltransferase (ChAT) enzyme activity of 3 nmol/mg protein/h; ChAT activity was blocked by 10 microM bromoacetylcholine. In conclusion, these data demonstrate the synthesis of the "neurotransmitter" acetylcholine in cultured glial cells, a finding which opens a new view upon the role of acetylcholine in mammalian brain.

Acetylcholine acts as a prominent transmitter in the central and peripheral nervous system. The aim of the present study was to investigate whether mammalian non-neuronal cells can synthesize and store acetylcholine. A cotton tipped applicator (Q-tip) was used to collect surface cells from airways and alimentary tract. Histological inspection indicated that rubbing of the luminal surface of human bronchi did not penetrate the basal membrane. Acetylcholine was measured by an HPLC-method using substrate-specific enzyme reactor-columns. Non-neuronal acetylcholine was found in cells covering inner and outer surfaces of rat and man. For example, acetylcholine was detected in the surface epithelium of human bronchi (33 pmol/g), mouth (female 0.7 and male 8 pmol/sample), small and large intestine (800 and 16 pmol/g, respectively), gall bladder (12 pmol/g), vagina (6 pmol/sample), skin 1000 (pmol/g) and in pulmonary pleura (5 pmol/sample). Somewhat higher amounts of acetylcholine were found in rat tracheal and intestinal epithelium and in rat skin. The synthesizing enzyme choline acetyltransferase (ChAT) was demonstrated in human surface epithelium by immunohistochemistry and by Western blot analysis. Enzymatic ChAT activity was demonstrated in isolated epithelial cells of human bronchi and small intestine (3.5 and 28 nmol/mg protein/h, respectively). Applied acetylcholine (in nM concentrations) increased, whereas inhibition of ChAT activity by bromoacetylcholine (10 microM) reduced the growth of cultured human bronchial epithelial cells. Inhibition of cell growth occurred also in the presence of atropine (1 microM) together with (+/-)-tubocurarine (30 microM). In conclusion, the present experiments demonstrate a widespread existence of non-neuronal acetylcholine in surface cells of man. Non-neuronal acetylcholine may act as a local signalling molecule.

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