Neurotensin receptors in canine intestinal smooth muscle: preparation of plasma membranes and characterization of (Tyr3-125I)-labelled neurotensin binding

To study the binding of (Tyr3-125I)-labelled neurotensin to intestinal muscle, plasma membranes have been purified from dog intestinal circular smooth muscle. Purification was done by differential centrifugation followed by separation on a sucrose gradient. Electron microscopic study revealed that the dissected circular muscles used as the source of membranes were free of myenteric plexus and that the plasma membrane fraction obtained was free of any mitochondria or synaptosomes. The fraction used was obtained at the interface of 14%-33% sucrose density on the gradient and was 25-times enriched in the plasma membrane marker enzyme 5'-nucleotidase activity as compared to post-nuclear supernatant. This fraction contained negligible activity of mitochondrial membrane marker enzyme cytochrome c oxidase and low activity of a putative endoplasmic reticulum marker enzyme NADPH-cytochrome-c reductase. This membrane fraction contained a high density of neurotensin binding sites. This binding was studied by kinetic and by saturation approaches. Analysis of data from saturation binding studies by the computer programs (EBDA and LIGAND) suggested the presence of a two-site model (Kd1 = 0.118 nM, Kd2 = 3.18 nM, Bmax1 = 9.73 fmol/mg and Bmax2 = 129.8 fmol/mg). A part of specifically bound neurotensin was rapidly dissociated. No cooperativity between the two receptor types could be detected. A kinetic analysis of binding gave the Kd value equal to 0.107 nM. Carboxy terminal amino acid residues 8-13 were found to be essential for the binding activity and replacement of Tyr11 by tryptophan reduced the affinity of the peptide by 10 times in displacement studies. Binding was modulated by sodium ions and a guanine nucleotide Gpp[NH]p. MgCl2, CaCl2 and KCl were also found to reduce the specific binding. Evidence was found of a high specific binding to another membrane fraction poor in plasma membranes and rich in synaptosomes. We concluded that plasma membrane of canine intestinal circular muscle contains neurotensin receptors with recognition properties distinct from those obtained in previous studies of neurotensin binding sites in murine tissues. Another neurotensin binding site may be present on neuronal membranes.     

Calcium and contractions of isolated smooth muscle cells from rat myometrium

Smooth muscle cells were isolated from estrogenized rat myometrium by collagenase digestion. Electron microscopic examination and measurement of cell lengths by image-splitting micrometry were carried out after fixation with acrolein. Mean lengths of cells before and after isolation were 81.7 and 66.9 micron, respectively. Responses of cells were compared with contractions of isolated strips recorded isometrically. Effects of carbachol and KCl were examined in 2 mM Ca, 2 mM Ca + 4 mM EGTA, and 2 mM Ca + 10(-8) M nitrendipine solution. Carbachol and KCl produced concentration-dependent shortening of isolated cells maximal at 30 s after addition. The concentrations of carbachol required to produce shortenings were about 100-fold less than those required to produce isometric contractions; but no major difference was observed in the concentration dependence of cell shortening and isometric contraction produced by potassium-induced depolarization. In 2 mM Ca solution, there was a phasic response, followed by a tonic response such that more than 50% of maximum cell shortening was maintained for 10 min. However, in 2 mM Ca + 4 mM EGTA or 10(-8) M nitrendipine, the tonic contraction was abolished and cells rapidly relaxed after 30 s. If carbachol was added to cells after varying times in the EGTA-containing solution, the ability to initiate a contraction declined exponentially with a half-time of 160 s. Effects of depolarization by KCl were examined in 2 mM Ca plus nitrendipine and 2 mM Ca + 4 mM EGTA solution. Shortening occurred in 2 mM Ca solution by depolarization but not if nitrendipine was added. Though shortening was not observed in 2 mM Ca + 4 mM EGTA solution by KCl, subsequent addition of carbachol induced shortening. These results suggested that there was an intracellular Ca store site from which Ca was released by carbachol and which was not affected by depolarization in the absence of external Ca. No evidence was obtained that the contraction persists in Ca2+-free medium in isolated cells, which is in agreement with previous findings in small muscle strips in which only a similar transient response was obtained.     

Intra-tree foraging behavior of Ceratitis capitata flies in relation to host fruit density and quality

We examined the intra-tree foraging behavior of individually-released, wild-population Mediterranean fruit flies (medflies), Ceratitis capitata (Wiedemann), on field-caged host trees bearing each of three different densities (0, 3, or 12 per tree) of non-infested host fruit (kumquat) or each of two levels of fruit quality (12 non-infested fruit or 12 fruit infested with eggs and covered with host marking pheromone). With increasing density of non-infested fruit, medflies tended to remain longer in trees, visit more fruit before leaving, oviposit more often, accept a proportionately smaller number of fruit visited, and emigrate sooner after the last egg was laid (i.e. have a shorter Giving-Up-Time). Medflies spent much less time, oviposited much less often, and exhibited a longer Giving-Up-Time on trees harboring pheromone-marked fruit than non-infested fruit. Variation in temperature within the range at which experiments were conducted (25–36°C) had little detectable influence on foraging behavior. We compare our findings with published findings on the intra-tree foraging behavior of another tephritid fly, Rhagoletis pomonella (Walsh), and with current foraging behavior theory. We discuss implications of our findings with respect to medfly management strategies, particularly fruit stripping in eradication programs and use of synthetic marking pheromone for control.

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Résumé Nous avons étudié le comportement de prospection dans un arbre, de femelles d'une population sauvage de C. capitata, libérées individuellement à l'intérieur de cages contenant des Eriobotrya japonica (kumquat), portant chacun 3 densités différentes de fruits no contaminés (0, 3, 12 par arbre) et chacun 2 niveaux de qualité de fruits: 12 fruits non infestés ou 12 fruits contaminés par des oeufs et recouverts de phéromone de marquage de l'hôte. C. capitata avait terndance à rester plus longtemps dans les arbres, à visiter plus de fruits avant le quitter, à pondre plus souvent, à accepter proportionnellement un nombre plus réduit de fruits déjà visités, à émigrer plus tôt après la ponte du dernier oeuf (c'est-à-dire à présenter un temps d'abandon plus bref), quand la densité des fruits non contaminés augmentait. C. capitata a dépensé beaucoup moins de temps, pondu beaucoup moins souvent, et présenté un temps d'abandon plus long sur les arbres portant des fruits marqués par la phéromone que sur ceux ayant des fruits non contaminés. Les variations de température dans la gamme de cells où les observations ont eu lieu (23–36°C) n'ont eu qu'une faible influence décelable sur le comportement de prospection. Nous avons comparé nos résultats avec ceux publiés sur la prospection à l'intérieur de l'arbre par une autre téphritide (Rhagoletis pomonella) et avec la théorie dominante sur le comportement de prospection. Nous discutons les conséquences de nos résultats sur les stratégies de lutte contre C. capitata, en particulier l'élimination des fruits dans les plans d'erradication et l'utilisation de phéromone synthétique de marquage.

     

Detection and localization of antigens on the surface of mouse spermatozoa. Antigen synthesis in the epididymis.

Spermatozoa achieve functional maturity during their transit through the epididymis and this maturation process is accompanied by changes in the composition and proteins of their surface. The addition of secretory products from the epididymis to the plasma membranes of the spermatozoa is considered to be a prerequisite for the acquisition by the spermatozoa of the capacities for forward motility and ovum recognition. An antibody was purified from an antiserum raised in the rabbit against fluid from the cauda epididymis of the mouse. This antibody, in combination with fluorescein isothiocyanate-conjugated goat anti-rabbit antibody, was used to demonstrate a progressive increase in the synthesis and secretion of antigens along the length of the epididymis. Immunoaffinity chromatography of [35S]methionine-labelled proteins, synthesized by segments of the epididymis maintained in vitro, showed that the predominant protein synthesized by the cauda, but not by the caput, epididymis, migrated on electrophoresis with an apparent Mr of 26,000. This same protein was the major antigen found on the plasma membrane of cauda spermatozoa that had been radioactively labelled with the non-penetrating probe isethionyl [1-14C]acetimidate.     

The projections of chemically identified nerve fibres in canine ileum

Summary The projections of nerve fibres with immunoreactivity for the peptides enkephalin (ENK), gastrin-releasing peptide (GRP), neuropeptide Y (NPY), somatostatin (SOM), substance P (SP) and vasoactive intestinal peptide (VIP) were studied in canine small intestine by analysing the consequences of lesions of intrinsic and extrinsic nerves. Of peptides present in fibres supplying myenteric ganglia, GRP, SOM and VIP were in anally directed nerve pathways, whereas ENK and NPY were in orally directed pathways. Pathways ran for up to about 30 mm. SP fibres ran for short distances in both directions in the myenteric plexus. The circular muscle was supplied with ENK, NPY, SP and VIP fibres arising from the myenteric ganglia, whereas most mucosal SP and VIP fibres were deduced to arise from submucous ganglia. There were projections of fibres reactive for ENK, GRP, SOM, SP and VIP from myenteric ganglia to submucous ganglia. Antibodies to tyrosine hydroxylase were used to locate noradrenaline nerve fibres supplying the intestine; these fibres all disappeared when extrinsic nerves running through the mesentery to the small intestine were cut. It is deduced that there is an ordered pattern of projections of peptide-containing fibres in the canine intestine.     

Characterization of quisqualate recognition sites in rat brain tissue usingDl-[3H]α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay

The binding of [³H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [³H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [³H]AMPA and [³H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K _d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K _d value=10 nM) without altering the number of binding sites (B _max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [³H]AMPA binding assay was quisqualate > AMPA > l-glutamate > kainate > d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP₇) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents.     

Presence of nucleosomes inPenicillium chrysogenum

We have studied the chromatin structure ofPenicillium chrysogenum. This fungus presents the typical nucleosomal repeat and the core DNA size characteristic of all the eukaryotes. The repeat length (about 180 base pairs) is in the range of those obtained for most fungi (160–180 base pairs) and shorter than in higher eukaryotes. Knowledge aboutP. chrysogenum chromatin structure opens the way to the study of the mechanisms of genetic regulation in this filamentous fungus.