High-affinity receptors for bombesin-like peptides in normal guinea pig lung membranes.

The binding of the radiolabelled bombesin analogue [125I-Tyr4]bombesin to guinea-pig lung membranes was investigated. Binding of [125I-Tyr4]bombesin was specific, saturable, reversible and linearly related to the protein concentration. Scatchard analysis of equilibrium binding data at 25 degrees C indicated the presence of a single class of non-interacting binding sites for bombesin (Bmax = 7.7 fmol/mg protein). The value of the equilibrium dissociation constant (KD = 90 pM) agrees with a high-affinity binding site. Bombesin and structurally related peptides such as [Tyr4]bombesin, neuromedin B and neuromedin C inhibited the binding of [125I-Tyr4]bombesin in an order of potencies as follows: [Tyr4]bombesin greater than bombesin greater than or equal to neuromedin C much greater than neuromedin B. These results indicate that guinea-pig lung membranes possess a single class of bombesin receptors with a high affinity for bombesin and a lower one for neuromedin B.     

Characterization of the cloned BamHI restriction modification system: its nucleotide sequence, properties of the methylase, and expression in heterologous hosts.

The BamHI restriction modification system was previously cloned into E. coli and maintained with an extra copy of the methylase gene on a high copy vector (Brooks et al., (1989) Nucl. Acids Res. 17, 979-997). The nucleotide sequence of a 3014 bp region containing the endonuclease (R) and methylase (M) genes has now been determined. The sequence predicts a methylase protein of 423 amino acids, Mr 49,527, and an endonuclease protein of 213 amino acids, Mr 24,570. Between the two genes is a small open reading frame capable of encoding a 102 amino acid protein, Mr 13,351. The M. BamHI enzyme has been purified from a high expression clone, its amino terminal sequence determined, and the nature of its substrate modification studied. The BamHI methylase modifies the internal C within its recognition sequence at the N4 position. Comparisons of the deduced amino acid sequence of M. BamHI have been made with those available for other DNA methylases: among them, several contain five distinct regions, 12 to 22 amino acids in length, of pronounced sequence similarity. Finally, stability and expression of the BamHI system in both E. coli and B. subtilis have been studied. The results suggest R and M expression are carefully regulated in a 'natural' host like B. subtilis.     

A fluorescent hydrophobic probe used for monitoring the kinetics of exocytosis phenomena

A fluorescence method is presented for quantitatively analyzing exocytosis phenomena and monitoring their kinetics. The method is based on the particular properties of a hydrophobic fluorescent probe, 1-[4-(trimethylammonio)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) [Prendergast, F.G., Haugland, R.P., & Callahan, P.J. (1981) Biochemistry 20, 7333-7338; Kuhry, J.G., Fonteneau, P., Duportail, G., Maechling, C., & Laustriat, G. (1983) Cell Biophys. 5, 129-140; Kuhry, J.G., Duportail, G., Bronner, C., & Laustriat, G. (1985) Biochim. Biophys. Acta 845, 60-67]. When this probe is interacted with intact resting cells in aqueous suspensions, it labels solely the membranes that are in contact with the external medium and is incorporated into them according to a partition equilibrium; i.e., the amount of the probe incorporated is proportional to the available membrane surface. TMA-DPH is highly fluorescent in membranes and not at all in water. Thus, a measurement of the TMA-DPH fluorescence intensity provides a signal proportional to the membrane surface. In secretory cells, the membrane surface available for the probe is increased upon fusion of the membrane of the secretory granules with the cell plasma membranes, directly or via intergranule fusion. Thus, when these cells are stimulated, more TMA-DPH is incorporated than in resting cells since the probe is allowed to also interact with the granule membranes now connected with the external medium by pores. This process results in a proportional increase in the TMA-DPH fluorescence intensity. The response was found to be very rapid and able to follow accurately the exocytosis kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Existence of very weak level of residual multiple innervation of Purkinje cells through climbing fibers in the cerebellum of the normal adult rat

Extra- or intracellular unit responses of Purkinje cells to the activation of climbing fibres were recorded in the cerebellum of adult rats. Out of 1.719 cels studied, only 4 were found to be innervated by more than one climbing fibre. This very small rate of residual multiple innervation in the normal adult illustrates the accuracy of the synaptic elimination process which leads to the innervation of each Purkinje cell through only one climbing fibre during normal development in the rat.     

Anaerobic Degradation of Cyanuric Acid, Cysteine, and Atrazine by a Facultative Anaerobic Bacterium

A facultative anaerobic bacterium that rapidly degrades cyanuric acid (CA) was isolated from the sediment of a stream that received industrial wastewater effluent. CA decomposition was measured throughout the growth cycle by using a high-performance liquid chromatography assay, and the concomitant production of ammonia was also measured. The bacterium used CA or cysteine as a major, if not the sole, carbon and energy source under anaerobic, but not aerobic, conditions in a defined medium. The cell yield was greatly enhanced by the simultaneous presence of cysteine and CA in the medium. Cysteine was preferentially used rather than CA early in the growth cycle, but all of the CA was used without an apparent lag after the cysteine was metabolized. Atrazine was also degraded by this bacterium under anaerobic conditions in a defined medium.     

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.     

Effects of cysteamine on pro-somatostatin related peptides

Intracerebroventricular (icv) injection of cysteamine to rats produced a marked depletion of somatostatin-14-like immunoreactivity (LI) in all rat brain regions examined. The somatostatin-28 (SS28)-LI and SS28(1-12)-LI were generally not altered by the cysteamine treatment. Following subcutaneous injection of the drug similar depletions of hypothalamic SS14-LI was observed with no change in SS28-LI nor SS28(1-12)-LI. In vitro cysteamine significantly increased the basal release of SS14-LI and markedly potentiated the evoked release of SS14-LI from hypothalamic slices. At 10 mM cysteamine, enhanced release of SS14-LI from hypothalamic slices was still observed despite a marked depletion of tissue content of SS14-LI.     

Accessory function of human thymic dendritic cells in Con A-induced proliferation of autologous thymocyte subsets.

Human thymic dendritic cells (DC) have previously been shown to be intimately associated with thymocytes in situ and in culture. We report that thymic DC express LFA-3 and ICAM-1 adhesion molecules and may spontaneously associate with autologous thymocytes within mitogen-independent clusters. Moreover, the accessory activity of isolated human thymic DC was investigated in Con A-stimulation assays. By proliferation experiments, measured as [3H]TdR incorporation, we demonstrated that irradiated thymic DC strongly increase the mitogen-induced activation of autologous PBL as well as of unfractionated thymocytes. More interestingly, in coculture assays performed with purified thymocyte subsets, we have found that thymic DC greatly enhance the Con A proliferation of CD1- CD3bright thymocytes whereas the accessory activity toward the CD1+ CD3- thymocytes was very weak. Inhibition experiments demonstrated that the DC accessory activity is inhibited by anti-DR-related and anti-IL-2R mAb. However, blocking assays with anti-CD11b, anti-CD11c, anti-LFA-3, and anti-ICAM1 mAb showed that the accessory function obtained is similar to that with untreated cultures. We conclude that isolated human thymic DC may present potent DR- and IL-2-dependent accessory activity mainly directed toward the CD1- CD3bright thymocyte subpopulation, suggesting that thymic DC may be involved in the in vivo proliferation of mature thymocytes.     

Activation and germination of Bacillus thuringiensis spores in Manduca sexta larval gut fluid

Incubation of Bacillus thuringiensis HD-1 spores in the larval gut fluid of Manduca sexta (tobacco hornworm) resulted in increased viable counts, conversion to phase-dark spores, and a loss of absorbance in spore suspensions, indicative of spore germination. Heat-activated and untreated spores incubated in water did not exhibit these changes. Only when spores were heat activated and incubated in germinants L-alanine and adenosine did changes in the spores approximate those observed in gut fluid. These data suggest that M. sexta larval gut fluid induces the activation and germination of B. thuringiensis spores.     

Identification of Brassica oleracea monosomic alien chromosome addition lines with molecular markers reveals extensive gene duplication

Summary Chromosomes of Brassica oleracea (2n=18) were dissected from the resynthesized amphidiploid B. napus Hakuran by repeated backcrosses to B. campestris (2n=20), creating a series of monosomic alien chromosome addition line plants (2n=21). Using morphological, isozyme and restriction fragment length polymorphism markers (RFLPs), 81 putative loci were identified. Of nine possible synteny groups, seven were represented in the 25 monosomic addition plants tested. Sequences homologous to 26% of the 61 DNA clones utilized (80% were cDNA clones) were found on more than one synteny group, indicating a high level of gene duplication. Anomalous synteny associations were detected in four 2n=21 plants. One of these plants showed two markers from one B. oleracea chromosome associated with a second complete B. oleracea synteny group, suggesting translocation or recombination between non-homologous chromosomes in Hakuran or the backcross derivatives. The other three 2n=21 plants each contained two or more B. oleracea synteny groups, suggesting chromosome substitution.