Effects of melanotan II,a melanocortin agonist,on grooming and exploration in rats after repeated restraint/immobilization

2 mg/kg melanotan II (MTII, administered i.p.), a cyclic peptide analog of α-melanocyte stimulating hormone, at a single dose increased grooming in naive rats placed in an unfamiliar open-field device without changing locomotion or rearing. Male rats exposed to restraint/immobilization stress (IS) for 1 h on three consecutive days displayed increased grooming after the second stressor exposure, compared to pre-stress levels. MTII, administered to the rats after IS, enhanced the grooming response compared both to the pre- and post-stress values. The increase was greatest after the first dose and declined over the following two applications. As to the locomotion of rats in the entire experimental space, IS reduced the distance moved only after the first two stressor exposures; MTII did not influence these alterations. Locomotion in the central part of arena was not reduced by the stressor or by MTII, on the contrary, there was an increase in both groups after the third intervention. The only observed change in rearing was an increase in the MTII group after the third restraint exposure. Thus, MTII selectively increased grooming without markedly affecting the spatio-temporal structure of locomotor behavior in the open-field. The decline of MTII enhanced grooming over the three test days may be interpreted in terms of adaptation to the stressor and of the developing tolerance to the peptide.     

Development of flow cytogenetics and physical genome mapping in chickpea (Cicer arietinum L.)

Procedures for flow cytometric analysis and sorting of mitotic chromosomes (flow cytogenetics) have been developed for chickpea (Cicer arietinum). Suspensions of intact chromosomes were prepared from root tips treated to achieve a high degree of metaphase synchrony. The optimal protocol consisted of a treatment of roots with 2mmol/L hydroxyurea for 18h, a 4.5-h recovery in hydroxyurea-free medium, 2h incubation with 10µmol/L oryzalin, and ice-water treatment overnight. This procedure resulted in an average metaphase index of 47%. Synchronized root tips were fixed in 2% formaldehyde for 20min, and chromosome suspensions prepared by mechanical homogenization of fixed root tips. More than 4×10⁵ morphologically intact chromosomes could be isolated from 15 root tips. Flow cytometric analysis of DAPI-stained chromosomes resulted in histograms of relative fluorescence intensity (flow karyotypes) containing eight peaks, representing individual chromosomes and/or groups of chromosomes with a similar relative DNA content. Five peaks could be assigned to individual chromosomes (A, B, C, G, H). The purity of sorted chromosome fractions was high, and chromosomes B and H could be sorted with 100% purity. PCR on flow-sorted chromosome fractions with primers for sequence-tagged microsatellite site (STMS) markers permitted assignment of the genetic linkage group LG8 to the smallest chickpea chromosome H. This study extends the number of legume species for which flow cytogenetics is available, and demonstrates the potential of flow cytogenetics for genome mapping in chickpea.     

Low frequency of BRCA1 germline mutations in 45 German breast/ovarian cancer families.

In this study we investigated 45 German breast/ovarian cancer families for germline mutations in the BRCA1 gene. We identified four germline mutations in three breast cancer families and in one breast-ovarian cancer family. among these were one frameshift mutation, one nonsense mutation, one novel splice site mutation, and one missense mutation. The missense mutation was also found in 2.8% of the general population, suggesting that it is not disease associated. The average age of disease onset in those families harbouring causative mutations was between 32.3 and 37.4 years, whereas the family harbouring the missense mutation had an average age of onset of 51.2 years. These findings show that BRCA1 is implicated in a small fraction of breast/ovarian cancer families suggesting the involvement of another susceptibility gene(s).     

Neuropeptide Y in the female reproductive tract of the rat. Distribution of nerve fibres and motor effects

The spontaneous firing of single neurones in the region of the lateral reticular nucleus was the subject of a pharmacological study employing microiontophoretic and systemic application of adrenoceptor agonists and antagonists. Both iontophoretic noradrenaline and systemic clonidine depressed neuronal firing. The depressions were consistently reversed by the alpha-2 antagonist RX781094. Other adrenergic antagonists, prazosin and sotalol, were ineffective. The results suggest the existence of alpha-2 receptors in this region of the brain.     

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.     

Isolation of a Trichoderma reesei cDNA encoding GTP: a-d-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation

A cDNA coding for GTP: α-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant. Received: 21 July 1997 / 24 April 1998     

Change in sympathetic activity,cardiovascular functions and plasma hormone concentrations due to cold water immersion in men

The purpose of this study was to determine whether or not repeated short-term cold water immersions can induce a change in the activity of the sympathetic nervous system and, consequently, in cardiovascular functions in healthy young athletes. Changes in some plasma hormone concentrations were also followed. A single cold water immersion (head-out, at 14°C, for 1 h) increased sympathetic nervous system activity, as evidenced by a four-fold increase (P < 0.05) in plasma noradrenaline concentration. Plasma adrenaline and dopamine concentrations were not increased significantly. Plasma renin-angiotensin activity was reduced by half (P < 0.05) during immersion but plasma aldosterone concentration was unchanged. Stimulation of the sympathetic nervous system during immersion did not induce significant changes in heart rate, but induced peripheral vasoconstriction (as judged from a decrease in skin temperature) and a small increase (by 10%) in systolic and diastolic blood pressures. No clear change in reactivity of the sympathetic nervous system was observed due to repeated cold water immersions (three times a week, for 6 weeks). Neither the plasma renin-angiotensin activity, aldosterone concentration nor cardiovascular parameters were significantly influenced by repeated cold water immersions. A lowered diastolic pressure and an increase in peripheral vasoconstriction were observed after cold acclimation, however. Evidently, the repeated cold stimuli were not sufficient to induce significant adaptational changes in sympathetic activity and hormone production.