Cell kinetic disturbances induced by treatment of human diploid fibroblasts with 5-azacytidine indicate a major role for DNA methylation in the regulation of the chromosome cycle

Summary BrdU-Hoechst flow cytometry was used to investigate the effects of DNA hypomethylation, induced by treatment with 5-azacytidine (5AC), on cell proliferation. When human fibroblast-like cells derived from skin and amniotic fluid were exposed to 5AC during three successive cell cycles, their clone-forming ability was diminished after removal of the drug. Treated cells were rendered quiescent by culture with low serum in the absence of the drug. Upon serum stimulation, they showed a diminished fraction of proliferating cells, which exhibited a prolonged transit through the S and G2 phase of the cell cycle, and a permanent arrest within the G2 compartment. This pattern of disturbed cell proliferation may in part explain the changes in replication banding pattern reported in the literature. Cytogenetic analysis of 5AC-treated cells revealed numerous endomitoses and tetraploid metaphases indicating a disturbed chromosome cycle in association with these cell kinetic perturbations.     

Addition of interleukin-2in vitro augments detection of lymphokine-activated killer activity generatedin vivo

Summary Thein vivo administration of repetitive weekly cycles of interleukin-2 (IL-2) to patients with cancer enhances the ability of freshly obtained peripheral blood lymphocytes (PBL) to lyse both the natural-killer(NK)-susceptible K562 and the NK-resistant Daudi targets. Lysis of both targets is significantly augmented by inclusion of IL-2 in the medium during the cytotoxicity assay. This boost is much greater for cells obtained following thein vivo IL-2 therapy than for cells obtained prior to the initiation of therapy or for cells from healthy control donors. In addition to direct lytic activity, the PBL obtained followingin vivo IL-2 show a rapid increase in lymphokine-activated killer (LAK) activity with more prolongedin vitro IL-2 exposure, indicating that LAK effectors primedin vivo respond with secondary-like kinetics to subsequent IL-2in vitro. Lymphocytes from healthy control individuals, cultured in IL-2 under conditions attempting to simulate thein vivo IL-2 exposure, function similarly to PBL obtained from patients following IL-2, in that low-level LAK activity was significantly boosted by inclusion of IL-2 during the cytotoxic assay and the cells also responded with secondary-like kinetics to subsequent IL-2in vitro. The augmentation of the LAK effect was also dependent on the dose of IL-2 added during the 4-h⁵¹Cr-release cytotoxicity assay, with higher doses of IL-2 having a more pronounced effect. While continuous infusion of IL-2 induces a greater cytotoxic potential per milliliter of blood obtained from patients, the peak serum IL-2 levels attained are greater with bolus IL-2 infusions. These pharmacokinetic results, together with the IL-2 dose dependence of LAK activity generatedin vivo shown in this report, suggest that a combination of treatment with bolus IL-2 infusions superimposed on continuous IL-2 infusion may transiently expose IL-2 dependent LAK cells, activatedin vivo, to higher concentrations of IL-2, facilitating theirin vivo cytotoxic potential.This work was supported by NIH contract NO1 CM-47669-02, NIH grants CA-32685, RR-031086, NO1 CM-47669-03, and American Cancer Society grant CH-237     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Distribution of glutamate decarboxylase-like immunoreactivity in the sixth abdominal ganglion of the cockroach Periplaneta americana

Summary An antiserum against glutamate decarboxylase (GAD) of the rat brain was used to locate GAD activity in sections of the nervous system of the cockroach, Periplaneta americana. The sixth abdominal ganglion was chosen because electrophysiological evidence suggests the presence of GABAergic inhibitory synapses in the cereal-giant interneuron system. Groups of somata and numerous fibres and tracts were positively labelled by the GAD antiserum. A posterior group of labelled somata could be identified close to the entry of the cereal nerves. A line of somata clusters lay along a ventro-lateral furrow. Another discrete row of GAD-like cells was located dorso-laterally. Some small cells among the dorsal unpaired neurons were labelled. A small central group appeared under these cells. An abundance of GAD-like processes and transversal tracts were found within the neuropile. The different systems of GABAergic inhibitors in the ganglion are discussed; in particular we show that the fibres of cereal nerve X are not labelled. This demonstrates that the latter act on the giant fibres via interneurons. We suggest that the group that sends axons into the overlapping region between the cereal nerve and the giant fibre could be the inhibitory interneurons involved in this system.     

Expression of the HNK-1/NC-1 epitope in early vertebrate neurogenesis

Summary A family of glycoconjugates has recently been shown to share a common carbohydrate epitope recognized by the mouse monoclonal antibody HNK-1. The specificity of HNK-1 was found to be similar to that of another monoclonal antibody, NC-1. These two IgM monoclonal antibodies were raised after immunization of mice with a human T-cell line and avian neural crest-derived ganglia, respectively. The antigens recognized by these antibodies include the myelin-associated glycoprotein, MAG, a glycolipid of defined structure, and a set of molecules involved in cell adhesion. The timing and pattern of appearance of these antigens are distinct. Moreover, the epitope may be absent on an antigen at a given stage or in a given tissue. Therefore, although the molecules able to carry the NC-1/ HNK-1 epitope are numerous and expressed in various tissues, the use of the monoclonal antibodies on tissue sections has proven adequate for following the migration of avian neural crest cells, the major cell lineage recognized by NC-1 and HNK-1 during early embryogenesis. Analogies in several other species have been found on the basis of HNK-1 reactivity. In this study we show that NC-1 and HNK-1 can be used successfully to label migrating neural crest cells in dog, pig and human. On the other hand, the NC-l/HNK-1 epitope was not present on migrating crest cells in amphibians or mice and was found only transiently on the neural crest of rats.     

Isolation and characterization of a thermophilic Methanobacterium able to use formate,the strain FTF

A thermophilic anaerobic which produced methane from formate and H₂ and CO₂ was isolated from a bench-scale digester treating a mixture of solid wastes at 55°C, after enrichment cultures on sodium acetate. The cells were slightly crooked rods occurring singly or in filaments. The bacterium was not motile, and stained Gram positive. Colonies appearing after 1 week of incubation were white with filamentous edges and 1 mm in diameter. The organism used H₂:CO₂ or formate as an energy source. Yeast extract was not required but stimulated growth significantly. Casamino acids were stimulatory and could serve as a nitrogen source. Cysteine was used as a sulfur source. The optimum pH for growth was 7.5. Growth occurred from 35 to 70°C with an optimum at 55°C. The deoxyribonucleic acid base composition was 49.2 mol% guanine plus cytosine. Though this isolate conforms to Methanobacterium thermoformicium, its proper assignment awaits further studies. It has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 3012.This work was supported in part by the Conseil Régional Nord/Pas-de-Calais     

Squid glycoproteins with structural similarities to Thy-1 and Ly-6 antigens

In an attempt to identify invertebrate homologs of Thy-1 antigen, the optic and central nervous tissue of squid was solubilized in deoxycholate and fractionated by lentil lectin affinity chromatography and gel filtration to yield small abundant glycoproteins. Material with biochemical similarities to Thy-1 was found and shown to consist of two glycoproteins that were ultimately purified using monoclonal antibody affinity columns. Both glycoproteins were sequenced to yield sequences of 84 residues for Sgp-1 and 92 residues for Sgp-2. The sequences were analyzed for similarities to Thy-1 and other Ig-related sequences, and Sgp-1 showed some similarities that were > 3 standard deviation units away from mean random scores when tested with the ALIGN program. However, the sequence patterns were not typical of Ig-related domains and the relationship of Sgp-1 to the Ig superfamily remains problematical. Sgp-2 showed no relationship to the Ig superfamily, but similarities to Ly-6 antigen sequences were noted that are in accord with an evolutionary relationship. The similarities included ten Cys residues in each sequence of which eight were matched in the best alignment given by the ALIGN program. Chemical evidence was obtained for glycophospholipid tails at the COOH-termini of Sgp-1 and Sgp-2 as is the case for Thy-1 and Ly-6 antigens.Abbreviations used in this paper GPL glycophospholipid - mAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.