Linkage studies with chromosome 17 DNA markers in 45 neurofibromatosis 1 families

A locus for von Recklinghausen neurofibromatosis (NF1) has recently been mapped near the chromosome 17 centromere. We have extended these linkage studies by genotyping 45 NF1 families with three DNA probes known to be linked to the chromosome 17 centromeric region. Of 34 families informative for NF1 and at least one of the three probes, 28 families show no recombinants with the disease gene. These data provide additional support for genetic homogeneity of NF1 and for a primary NF1 locus linked to the chromosome 17 centromere. Among the informative families were 7 families with apparent new NF1 mutations. Our data suggest that these mutations are probably at the chromosome 17 NF1 locus.     

A new mutant class of soybean lacks urease in leaves but not in leaf-derived callus or in roots

Summary We reported earlier the recovery of two classes of soybean urease mutants in soybean (Glycine max L. Merr. cv. Williams). Class I mutants lack the embryo-specific urease while class II mutants lack the activities of both urease isozymes, the embryo-specific and the ubiquitous urease, the latter found in all tissues examined. We report here the recovery of a true-breeding mutant, aj3, which represents the third phenotypic class: normal levels of embryo-specific urease and little or no ubiquitous urease. Unlike class II mutant plants which lack urease in all tissue, aj3 lacks urease activity only in leaves (ca. 2% normal activity); its roots have near normal urease activity. Callus derived from leaves of aj3 has 14% to 40% the urease activity of Williams 82 callus. This partial reduction in urease activity in aj3 callus is sufficient to reduce growth with urea as sole nitrogen source and to confer resistance to 50 mM urea added to callus maintenance medium. Leaves of aj3 produce more than 40 times the urease antigen expected from their urease activity. The aj3 trait is due to a single recessive lesion which is not allelic with lesions at theEu2, Eu3 (class II) orEu1 (class I) loci. We designate the aj3 genotype aseu4/eu4.     

Fatty acid metabolism in hepatocytes cultured with hypolipidaemic drugs. Role of carnitine.

The kinetic features of the changes in the cytosolic free Ca2+ concentration, [Ca2+]i, following stimulation by thyrotropin releasing hormone (TRH) were analysed in single cells of a pituitary line (GH3B6) by dual excitation microfluorimetry [Tsien, Rink & Poenie (1985) Cell Calcium 6, 145-157], using fura 2 as intracellular Ca2+ probe. Two phases were observed: initially, [Ca2+]i is raised in a single rapid transient to a maximum averaging 8.0 microM, and in a second phase TRH causes a series of rapid [Ca2+]i oscillations with maxima around 1.0 microM, which are probably due to the enhanced firing of action potentials. TRH triggers both phases independently, i.e. it can elicit either the first or the second phase exclusively. This is also the case in those cells in which [Ca2+]i undergoes rhythmic oscillations due to the firing of spontaneous action potentials [Schlegel, Winiger, Mollard, Vacher, Wuarin, Zahnd, Wolheim & Dufy (1987) Nature (London) 329, 719-721]. The sudden onset of the first phase of TRH action on [Ca2+]i shows that Ca2+ mobilization due to enhanced production of inositol phosphate may occur as rapidly as the firing of action potentials, i.e. in the ms time range. Due to a marked response type heterogeneity and to the randomness of the rapid events, previous monitoring of [Ca2+]i in cell populations had misleadingly suggested small and maintained changes due to TRH. It is concluded that stimulatory regulation of secretion is provided by the generation of rapid [Ca2+]i transients, the frequency of which determines secretory rate. Furthermore, it is demonstrated that the regulation of [Ca2+]i by hormones and neurotransmitters in pituitary and many other cell types will have to be studied at the single cell level in order to appreciate its role in cell activation.     

Neural function: metabolism and actions of inositol metabolites in mammalian brain

In the nervous system, a variety of cell types respond to external stimuli through the inositol lipid signalling pathways. The stimulus-coupled sequence of intracellular events has been investigated in a homogeneous model system, the cloned mammalian neural cell line NG115-401L. The neural peptide bradykinin stimulates a rapid production of identified inositol phosphate isomers and an intracellular Ca2+ discharge followed by a persistent plasma membrane influx. The temporal sequence suggests that Ins(1,4,5)P3 or Ins(1,3,4,5)P4 or both may coordinate these events in a neuronal cell, as has been suggested in other cell types. Thapsigargin, an irritant and tumour-promoting plant product, produces calcium transients in the absence of inositol phosphate production, and may provide a new tool for investigating the interactions between inositol phosphates and changes in cellular calcium homeostasis. In the 401L line, high levels of radiolabelled InsP5 and InsP6 have been detected, which has led to the evaluation of their possible occurrence and actions in normal brain. Both InsP5 and InsP6 are produced from a radiolabelled myo-inositol precursor in intact mature brain in a region-specific manner. This suggests that both inositol polyphosphates may be end products of regionally regulated biosynthetic pathways. When microinjected into a nucleus of the brainstem, or iontophoretically applied to the dorsal horn of the spinal cord, both InsP5 and InsP6, but not Ins(1,3,4,5)P4 isomers, appear to be potent neural stimulants. These results suggest that the inositol lipid signalling pathways may generate both intracellular and extracellular signals in brain.     

The isolation and immunolocalization of iron-binding compounds produced by Gloeophyllum trabeum

Summary Low molecular weight iron-binding compounds are produced by the brown-rot fungus Gloeophyllum trabeum. These chelators may function in scavenging transition metals for fungal metabolism and extracellular enzyme production. Because of the low molecular mass of the chelate-metal complex (below 1000 Da), and the oxidizing potential of the bound transition metals, certain chelating compounds could also play a role in the early stages of cellulose depolymerization by brown-rot fungi. High-affinity iron-binding compounds were isolated and partially purified from both liquid cultures of the brown-rot Gloeophyllum trabeum and from infected wood. Chelating compounds purified by thin-layer chromatography were used to prepare specific antibodies. These antibodies were shown to detect the chelator in infected wood and liquid fungal cultures by enzyme-linked immunosorbent assay and could be used in immunotransmission electron microscopy to visualize the high-affinity iron-binding compounds in situ. Elucidating the physiological roles of fungal chelate-metal complexes and determining their function in lignocellulose depolymerization will help us to better understand the mechanism of wood biodegradation.Publication no. 1549 Maine Agricultural Experiment Station Offprint requests to: J. Jellison     

Fatty acid uptake and metabolism to ketone bodies and triacyglycerol in rat and human hepatocyte cultures is dependent on chain-length and degree of saturation. Effects of carnitine and glucagon

Rat and human hepatocyte cultures were incubated with 5 common plasma longchain fatty acids (C16-C18). Rates of fatty acid uptake were similar in rat and human hepatocytes and were of the order: 16:1 greater than 16:0; 18:2 greater than 18:1 greater than 18:0. Rates of ketogenesis were lower in human compared to rat hepatocytes. In rat hepatocytes glucagon stimulated ketogenesis only in the presence of exogenous carnitine and rates of ketogenesis were higher from unsaturated compared to corresponding saturated fatty acids. Glucagon decreased triacylglycerol secretion irrespective of the fatty acid substrate and it increased intracellular triacylglycerol accumulation. The latter effect of glucagon was more marked in the absence of carnitine supplementation.     

Effect of insulin on ketogenesis and fatty acid synthesis in rat hepatocytes incubated with dichloroacetate

In parenchymal liver cells isolated from fed rats, insulin increased the formation of 14CO2 from [1-14C]pyruvate (and presumably the flux through pyruvate dehydrogenase) by 14%. Dichloroacetate, an activator of the pyruvate dehydrogenase complex, stimulated this process by 133%. As judged from the conversion of [2-14C]pyruvate to 14CO2, the tricarboxylic acid cycle activity was not affected by insulin, but it was depressed by dichloroacetate. In hepatocytes from fed rats, incubated with glucose as the only carbon source, dichloroacetate caused a stimulation (31%) of fatty acid synthesis, measured as 3H incorporation from 3H2O into fatty acid, and an increased (134%) accumulation of ketone bodies (acetoacetate + D-3-hydroxybutyrate). Dichloroacetate did not affect ketone body formation from [14C]palmitate, suggesting that the increased accumulation of ketone bodies resulted from acetyl-CoA derived from pyruvate. Insulin stimulated fatty acid synthesis in hepatocytes from fed rats. In the combined presence of insulin plus dichloroacetate, fatty acid synthesis was more rapid than in the presence of either insulin or dichloroacetate, whereas the accumulation of ketone bodies was smaller than in the presence of dichloroacetate alone. Although pyruvate dehydrogenase activity, which is rate-limiting for fatty acid synthesis in hepatocytes from fed rats, is stimulated both by insulin and by dichloroacetate, the reciprocal changes in fatty acid synthesis and ketone body accumulation brought about by insulin in the presence of dichloroacetate suggest that insulin is also involved in the regulation of fatty acid synthesis at a mitochondrial site after pyruvate dehydrogenase, possibly at the partitioning of acetyl-CoA between citrate and ketone body formation.     

Observations of Barophilic Microbial Activity in Samples of Sediment and Intercepted Particulates from the Demerara Abyssal Plain

To better understand the ecological significance of pressure effects on bacteria in the abyssobenthic boundary layer, experimental suspensions of sediments and sinking particulates were prepared from samples collected in boxcore and bottom-moored sediment traps at two stations (depth, 4,470 and 4,850m) in the Demerara abyssal plain off the coast of Brazil. Replicate samples were incubated shipboard at 3°C and at both atmospheric and deep-sea pressures (440 or 480 atm [4.46 × 10⁴ or 4.86 × 10⁴ kPa]) following the addition of [¹⁴C]glutamic acid (<10 μg liter⁻¹) or yeast extract (0.025%) and the antibiotic nalidixic acid (0.002%). In seven of the eight samples supplemented with isotope, a barophilic microbial response was detected, i.e., substrate incorporation and respiration were greater under in situ pressure than at 1 atm (101.3 kPa). In the remaining sample, prepared from a sediment trap warmed to 24°C before recovery, pressure was observed to inhibit substrate utilization. Total bacterial counts by epifluorescence microscopy decreased with depth in each sediment core, as did utilization of glutamic acid. Significant percentages of the total bacterial populations in cold sediment trap samples (but not the prewarmed one or any boxcore sample) were abnormally enlarged and orange fluorescing after incubation with yeast extract and nalidixic acid under deep-sea conditions. Results indicated that in the deep sea, barophilic bacteria play a predominant role in the turnover of naturally low levels of glutamic acid, and the potential for intense microbial activity upon nutrient enrichment is more likely to occur in association with recently settled particulates, especially fecal pellets, than in buried sediments.