Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Spawning behaviour ofCheimerius nufar in captivity

Synopsis The santerCheimerius nufar is widespread in tropical and subtropical waters of the western Indian Ocean and forms an important component of linefish catches along the east coast of southern Africa. Observations of spawning behaviour in captivity have revealed that spawning occurs during spring over a period of four months. Mating takes place at sunrise and may continue for up to 105 min (mean duration = 60 min). During spawning males become dark with prominent white markings. They become very aggressive and set up territories. Females remain a uniform silvery-pink. Mating occurs between males and females of similar sizes, culminating in egg and sperm release near the surface. Individual fish spawned up to 14 times during a morning. Streaking occurred throughout the season, with a second male joining a spawning pair and releasing gametes simultaneously. Slinger,Chrysoblephus puniceus, a dominant fish on offshore reefs in the same region, interfered with spawning throughout the season and were observed to eat eggs when they were released. The spawning strategy ofC. nufar is similar to protogynous species in several respects, indicating that this functional gonochorist may not conform to current theoretical predictions.     

Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants

Extractive fermentation has been proposed to enhance the productivity of fermentations that are end product inhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed to circumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated the inhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlled conditions. The production of acetone and acetoin was also increased, even though these two products were not extractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementation of glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement of butanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was not observed unless an organic phase was present to extract butanol. A mechanism for the effects of decanol on product formation is proposed.     

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)- propylsulfonyl]-gamma-oxo-benzenebutanoate: a leukotriene D4 receptor antagonist

L-648,051, sodium 4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy) propylsulfonyl]-gamma-oxo-benzenebutanoate is a selective and competitive inhibitor of [3H]leukotriene D4 (KB value of 4.0 microM) and to a lesser extent [3H]leukotriene C4 (Ki value of 36.7 microM) binding in guinea pig lung homogenates. Functionally, it selectively antagonized contractions of guinea pig trachea induced by leukotrienes C4, D4, E4, and F4 in concentrations that did not antagonize contractions induced by acetylcholine, histamine, serotonin, prostaglandin F2 alpha, or U-44069 (endoperoxide analogue). Schild plot analysis indicated that L-648,051 competitively antagonized contractions of guinea pig ileum induced by leukotriene D4 (pA2 7.7) and contractions of trachea induced by leukotrienes D4, E4, and F4 (pA2 7.3, 7.4, and 7.5, respectively). Contractions of guinea pig trachea induced by leukotriene C4 were inhibited in a noncompetitive fashion (Schild plot slope, 0.45). Developed contractions of trachea induced by the leukotrienes were rapidly reversed by L-648,051 greater than FPL-55712 greater than L-649,923. Intravenous L-648,051 selectively blocked bronchoconstriction induced in anaesthetized guinea pigs by intravenous leukotrienes C4, D4, and E4 but not that induced by arachidonic acid, serotonin, U-44069, or acetylcholine. The compound displayed poor activity following intraduodenal administration. The profile of activity for L-648,051 indicates that it may be a useful topical agent for studying the role of leukotrienes in diseases such as bronchial asthma.     

Secondary production of an intertidal mussel (Mytilus edulis L.) population in the Eastern Scheldt (S.W. Netherlands)

We monitored an intertidal mussel (Mytilus edulis L.) population between June 1981 and June 1982 in the Eastern Scheldt estuary (S.W. Netherlands). Density and biomass of the population remained relatively constant over the study period. The shell length growth was described by a Gompertz growth curve. The parameters of this equation were estimated from a log-log-modified Ford-Walford plot of the growth-ring data. The slope of the relationship between animal weight and shell length is season-dependent, mainly due to the spawning cycle in larger mussels.Secondary production is estimated with the growth rate method. In the calculated growth rates the change in slope of the length-weight relationship is incorporated, as well as differences in length growth rates between summer and winter. Secondary production amounts to 156 g AFDW m^–2a ^–1 (expressed per m² of mussel bank). P:B is 0.50 a^–1. The mussel productivity is probably a limiting factor for the density of overwintering Oystercatchers (Haematopus ostralegus).     

Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: I. Effects of Dopamine Precursors, Reserpine, Amphetamine, and l-DOPA Decarboxylase and Monoamine Oxidase Inhibitors

The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenylethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydroxyphenylalanine (L-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, L-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous L-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors pargyline and (-)-deprenyl differentially affected the degradation of DA, since 100 microM pargyline was apparently more effective than 100 microM (-)-deprenyl. Reserpine and (+/-)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (+/-)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.     

Virulence genes are carried by a megaplasmid of the plant pathogen Pseudomonas solanacearum

Summary A class of avirulent mutants of the plant pathogenic bacterium Pseudomonas solanacearum, strain GMI1000, resistant to acridine orange (Acr^r), harbour a deletion of over 85 kb in their genome. This deletion affects, a1,000 kb megaplasmid which has previously been shown to be present in most of the strains of this species. In addition at least 11 out of 13 independent Tn5 insertions, leading to loss of virulence, are located on the megaplasmid. Nine of them are present in the region which is deleted from the Acr^r mutants. These results suggest that the majority of virulence genes identified so far are plasmid borne.     

Growth Regulators Have Rapid Effects on Photosynthate Unloading from Seed Coats of Phaseolus vulgaris L

Of nine plant growth regulators (indoleacetic acid, 1-naphthalene acetic acid, gibberellic acid, giberellin 4/7, 6-benzylaminopurine, 6-furfurylaminopurine, abscisic acid, and 1-aminocyclopropane carboxylic acid) tested, only 6-benzylaminopurine and abscisic acid affected ¹⁴C-photosynthate unloading from excised seed coats of Phaseolus vulgaris L. Unloading, in the presence of KCl, was stimulated by 25 to 40%. Stimulation occurred immediately for 6-benzylaminopurine and for abscisic acid within 10 to 12 minutes of application.     

Dipyridamole-insensitive nucleoside transport in mutant murine T lymphoma cells

From a mutagenized population of S49 murine T lymphoma cells, a mutant cell line, JPA4, was selected that expressed an altered nucleoside transport capability. JPA4 cells transported low concentrations of purine nucleosides and uridine more rapidly than the parental S49 cell line. The transport of these nucleosides by mutant cells was insensitive to inhibition by either dipyridamole (DPA) or 4-nitrobenzylthioinosine (NBMPR), two potent inhibitors of nucleoside transport in mammalian cells. Kinetic analyses revealed that the apparent Km values for the transport of uridine, adenosine, and inosine were 3-4-fold lower in JPA4 cells compared to wild type cells. In contrast, the transport of both thymidine and cytidine by JPA4 cells was similar to that of parental cells, and transport of these pyrimidine nucleosides remained sensitive to inhibition by both NBMPR and DPA. Furthermore, thymidine was a 10-12-fold weaker inhibitor of inosine transport in JPA4 cells than in wild type cells. Thus, JPA4 cells appeared to express two types of nucleoside transport activities; a novel (mutant) type that was insensitive to inhibition by DPA and NBMPR and transported purine nucleosides and uridine, and a parental type that retained sensitivity to inhibitors and transported cytidine and thymidine. The phenotype of the JPA4 cell line suggests that the sensitivity of mammalian nucleoside transporters to both NBMPR and DPA can be genetically uncoupled from its ability to transport certain nucleoside substrates and that the determinants on the nucleoside transporter that interact with each nucleoside are not necessarily identical.     

Physicochemical characterization of human fibroblast migration inhibitory factor

We recently reported a new lymphokine activity that affects fibroblasts by inhibiting their spontaneous migration. Human fibroblast migration inhibitory factor (FIF) obtained from concanavalin A (Con A)-stimulated human lymphocytes was characterized by Sephadex gel filtration and by enzyme treatment. FIF was found to be stable at 56 degrees C for 15 min but destroyed at 80 degrees C or at pH lower than 5. Gel filtration revealed two peaks of FIF activity 15,000 and at 34,000 Da. FIF activity was lost following treatment with trypsin, chymotrypsin, and neuraminidase and FIF could not be generated in the presence of inhibitors of glycosylation, suggesting that the molecule was a glycoprotein. FIF could be removed by adsorption to human fibroblasts but not to PMN, monocytes, or red blood cells. Further studies were carried out to investigate the role of sugars in the interaction of FIF with the target cells. Human FIF activity was significantly reduced in the presence of several sugars including alpha-methyl-D-mannoside, L-xylose, N-acetyl-D-glucosamine, D-mannose, L-rhamnose but not L-fucose. Preincubation of human fibroblasts with alpha-methyl-D-mannoside prevented their response to FIF. In contrast, pretreatment of fibroblasts with mannosidase had no effect, suggesting that alpha-methyl-D-mannoside was an essential component of the FIF molecule recognized by the FIF receptor on fibroblasts.