Determining relative abundance of specific DNA sequences in flow cytometrically sorted maize nuclei

A wide breadth of DNA content variation has been reported amongmaize lines. While the extent of this variation has been welldocumented, few studies have focused on its cause. Some of thenuclear DNA content variation has been explained by the presenceof B chromosomes or knobs. However, variation in these two structuresdoes not account for all of the observed variation. In orderto identify other fluctuating DNA sequences, a rapid and reliablemethod of estimating relative abundance of DNA sequences neededto be developed. The potential of flow cytometry in conjunctionwith spot hybridization for determining relative abundance ofspecific DNA sequences in maize was studied. Different numbersof G1 phase nuclei were sorted on nitrocellulose filters andnon-radioactive hybridization and signal detection performed.Results from these experiments revealed a significant, positivelinear correlation between the amount of target sequence andsignal density using both knob (R = 0.98) and ribosomal spacer(R =0.99) DNA sequences. In addition, G1 phase nuclei of eightinbred lines differing in the amount of knob heterochromatin,were sorted on to filters and the non-radioactive hybridizationand signal detection performed. A significant, positive linearcorrelation between C-band number and signal density (R =0.83;P = 0.0051) as well as between per cent heterochromatin andsignal density (R=0.96;P = 0.0002) was observed. These resultsindicate the usefulness of flow cytometry for spot hybridizationin determining the relative abundance of DNA sequences in themaize genome. Key words: Flow cytometry, copy number, non-isotope labelling, spot hybridization, flow sorting, Zea mays L.     

Polyamine derivatives as inhibitors of trypanothione reductase and assessment of their trypanocidal activities

Trypanothione reductase (TR) occurs exclusively in trypanosomes and leishmania, which are the etiological agents of many diseases. TR plays a vital role in the antioxidant defenses of these parasites and inhibitors of TR have potential as antitrypanosomal agents. We describe the syntheses of several spermine and spermidine derivatives and the inhibiting effects of these compounds on T. cruzi TR. All of the inhibiting compounds displayed competitive inhibition of TR-mediated reduction of trypanothione disulfide. The three most effective compounds studied were N⁴,N⁸-bis(3-phenylpropyl)spermine (12), N⁴,N⁸-bis(2-naphthylmethyl)spermine (14), and N¹,N⁸-bis(2-naphthylmethyl)spermidine (21), with K_i values of 3.5, 5.5 and 9.5 μM, respectively. Compounds 12, 14, and 21 were found to be potent trypanocides in vitro with IC₅₀ values ranging from 0.19 to 0.83 μM against four T. brucei ssp. strains. However, these compounds did not prolong the lives of mice infected with trypanosomes. This work indicates that certain polyamine derivatives which target a unique pathway in Trypanosomatidae have potential as antitrypanosomal agents.     

Characterization of the Palmitoylation Domain of SNAP-25

Abstract: SNAP-25 (synaptosomal associated protein of 25 kDa) is a neural specific protein that has been implicated in the synaptic vesicle docking and fusion process. It is tightly associated with membranes, and it is one of the major palmitoylated proteins found in neurons. The functional role of palmitoylation for SNAP-25 is unclear. In this report, we show that the palmitate of SNAP-25 is rapidly turned over in PC12 cells, with a half-life of ∼3 h, and the half-life for the protein is 8 h. Mutation of Cys to Ser at positions 85, 88, 90, and 92 reduced the palmitoylation to 9, 21, 42, and 35% of the wild-type protein, respectively. Additional mutations of either Cys^85,88 or Cys^90,92 nearly abolished palmitoylation of the protein. A similar effect on membrane binding for the mutant SNAP-25 was observed, which correlated with the degree of palmitoylation. These results suggest that all four Cys residues are involved in palmitoylation and that membrane association of SNAP-25 may be regulated through dynamic palmitoylation.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

mRNA translocation and microtubules: insect ovary models

The nurse cells in insect ovarioles supply the developing oocytes with various cellular components, including mRNAs, which pass from one cell to the other through intercellular bridges traversed by microtubules. Best studied of these mRNAs are those that encode the axis-determining factors in Drosophila embryos. These mRNAs are further translocated and localized within the oocyte to sites where the products of their translation will ultimately function. This article explores the evidence supportive of a role for microtubules and motor proteins in these processes.     

Lipogenesis and the synthesis and secretion of very low density lipoprotein by avian liver cells in nonproliferating monolayer culture. Hormonal effects

The nonproliferating chicken liver cell culture system described yields cell monolayers with morphological and lipogenic properties characteristic of the physiological-nutritional state of donor animals. Synthesis and secretion of fatty acid, cholesterol, and very low density lipoprotein (VLDL) occur at in vivo rates and respond to hormones and agents which affect these processes in vivo. Cells derived from fed chickens maintain high rates of synthesis of fatty acid and cholesterol for several days if insulin is present in the medium. High rates of fatty acid synthesis are correlated with the appearance of membrane-enclosed triglyceride-rich vesicles in the cytoplasm; deletion of insulin causes a decrease (T1/2 = 22 h) in fatty acid synthetic activity. Addition of glucagon or cyclic AMP (cAMP) causes an immediate cessation of fatty acid synthesis and blocks the appearance of the triglyceride-rich vesicles. Fatty acid synthesis in liver cells prepared from fasted chickens is less than 5% that of cells from fed animals. After 2-3 days in culture with serum-free medium containing insulin +/- triiodothyronine, fatty acid synthesis is restored to normal; glucagon or dibutyryl cAMP blocks this recovery. Liver cells derived from estradiol-treated chickens synthesize and secrete VLDL for at least 48 h in culture. Electron micrographs of these cells reveal more extensive development of the rough endoplasmic reticulum and Golgi complex compared to cells from untreated chickens. Whereas [3H]leucine incorporation into total protein is unaffected by estrogen treatment, [3H]leucine incorporation into cellular and secreted immunoprecipitable VLDL is markedly increased indicating specific activation of VLDL apopeptide synthesis; 8-10% of the labeled protein synthesized and secreted is VLDL. Dodecyl sulfate-acrylamide gel electrophoresis of immunoprecipitated 3H-VLDL reveals three major apopepetides of 300,000, 11,000, and 8,000 daltons corresponding to those of purified chicken VLDL.     

Glucose-6-phosphate dehydrogenase porbandar: A new slow variant with slightly reduced activity in a South African family of Indian descent

A new variant of the red cell enzyme glucose-6-phosphate dehydrogenase has been detected in a South African male of Indian descent and in several of his relatives. The enzyme variant is characterized by slow electrophoretic mobility, low Michaelis constants for the substrates glucose-6-phosphate and NADP, and increased utilization of the substrate analogues 2-deoxyglucose-6-phosphate and deamino-NADP relative to the normal (B⁺) enzyme. There is no evidence that the enzyme variant, for which the name G6PD Porbandar is suggested, is associated with any hematological abnormality.The Atomic Energy Board and the South African Medical Research Council provided support for part of this work.     

Platte Valley Yellows,a chlorotic condition of soybeans: Symptoms and preliminary chemical analyses

Summary A chlorotic condition of soybeans in the river valleys of eastern Nebraska was studied. The pattern of chlorosis, presence of petiolar necrosis, reductions in rhizobial and mycorrhizal infection, and plant and soil chemical analyses suggest that the condition is more complex than normal iron deficiency chlorosis or other mineral deficiencies or toxicities. Nodulation is almost totally inhibited in affected plants. Mycorrhizal fungi, while able to infect early in the season, do not persist on affected plants. Breakdown of endodermis was observed in the roots of affected plants. The factors contributing to this condition have not yet been determined. Published with the approval of the director as Paper No. 5672, Journal Series, Nebraska Agricultural Experiment Station.