Beneficial effects of activated charcoal on bulblet production in tissue cultures of Muscari armeniacum

Production of bulblets of Muscari armeniacum through tissue culture is enhanced when 1 g/l activated charcoal is added to a modified Murashige and Skoog (MS) medium. Bulblet regeneration is direct from bulb scale explants with no intermediate callus growth. Bulblets can be transferred successfully to a greenhouse environment directly from aseptic culture.     

Methylammonium uptake by Rhodobacter capsulatus

The ammonium uptake system of Rhodobacter capsulatus B100 was examined using the ammonium analog methylammonium. This analog was not transported when cells were grown aerobically on ammonium. When cultured on glutamate as a nitrogen source, or when nitrogen-starved, cells would take up methylammonium. Therefore, in cells grown under nitrogen-limiting conditions, a second system of ammonium uptake (or a modified form of the first) is present which is distinguished by its capacity for transporting the analog in addition to ammonium. The methylammonium uptake system exhibited saturation kinetics with a K _m of 22 M and a V _max of about 3 nmol per min · mg protein. Ammonium completely inhibited analog transport with a K _i in the range of 1 M. Once inside the cell methylammonium was rapidly converted to -N-methylglutamine; however, a small concentration gradient of methylammonium could still be observed. Kinetic parameters reflect the effects of assimilation.The methylammonium uptake system was temperature and pH dependent, and inhibition studies indicated that energy was required for the system to be operative. A glutamine auxotroph (G29) lacking the structural gene for glutanime synthetase did not accumulate the analog, even when nitrogen starved. The Nif^- mutant J61, which is unable to express nitrogenase structural genes, also did not transport methylammonium, regardless of the nitrogen source for growth. However, the mutant exhibited wild-type ammonium uptake and glutamine synthetase activity. These data suggest that transport of ammonium is required for growth on limited nitrogen and is under the control of the Ntr system in R. capsulatus.Abbreviations CCCP carbonyl cyanide-m-chlorophenyl hydrazone - CHES cyclohexylaminoethanesulfonic acid - DMSO dimethyl sulfoxide - GMAD -N-methylglutamine - GS glutamine synthetase - MES 2-(N-morpholino) ethanesulfonic acid - MSX methionine-Dl-sulfoximine - pCMB p-chloromercuribenzoate - Tricine N-tris(hydroxymethyl)methylglycine     

Glycopeptides of the Type-Common Glycoprotein gD of Herpes Simplex Virus Types 1 and 2

We have carried out detailed structural studies of the glycopeptides of glycoprotein gD of herpes simplex virus types 1 and 2. We first examined and compared the number of N-asparagine-linked oligosaccharides present in each glycoprotein. We found that treatment of either pgD-1 or pgD-2 with endo-β-N-acetylglucosaminidase H (Endo H) generated three polypeptides which migrated more rapidly than pgD on gradient sodium dodecyl sulfate-polyacrylamide gels. Two of the faster-migrating polypeptides were labeled with [³H]mannose, suggesting that both pgD-1 and pgD-2 contained three N-asparagine-linked oligosaccharides. Second, we characterized the [³H]mannose-labeled tryptic peptides of pgD-1 and pgD-2. We found that both glycoproteins contained three tryptic glycopeptides, termed glycopeptides 1, 2, and 3. Gel filtration studies indicated that the molecular weights of these three peptides were approximately 10,000, 3,900, and 1,800, respectively, for both pgD-1 and pgD-2. Three methods were employed to determine the size of the attached oligosaccharides. First, the [³H]mannose-labeled glycopeptides were treated with Endo H, and the released oligosaccharide was chromatographed on Bio-Gel P6. The size of this molecule was estimated to be approximately 1,200 daltons. Second, Endo H treatment of [³⁵S]methionine-labeled glycopeptide 2 reduced the molecular size of this peptide from approximately 3,900 to approximately 2,400 daltons. Third, glycopeptide 2 isolated from the gD-like molecule formed in the presence of tunicamycin was approximately 2,200 daltons. From these experiments, the size of each N-asparagine-linked oligosaccharide was estimated to be approximately 1,400 to 1,600 daltons. Our experiments indicated that glycopeptides 2 and 3 each contained one N-asparagine-linked oligosaccharide chain. Although glycopeptide 1 was large enough to accommodate more than one oligosaccharide chain, the experiments with Endo H treatment of the glycoprotein indicated that there were only three N-asparagine-linked oligosaccharides present in pgD-1 and pgD-2. Further studies of the tryptic glycopeptides by reverse-phase high-performance liquid chromatography indicated that all of the glycopeptides were hydrophobic in nature. In the case of glycopeptide 2, we observed that when the carbohydrate was not present, the hydrophobicity of the peptide increased. The properties of the tryptic glycopeptides of pgD-1 were compared with the properties predicted from the deduced amino acid sequence of gD-1. The size and amino acid composition compared favorably for glycopeptides 1 and 2. Glycopeptide 3 appeared to be somewhat smaller than would be predicted from the deduced sequence of gD-1. It appears that all three potential glycosylation sites predicted by the amino acid sequence are utilized in gD-1 and that a similar number of glycosylation sites are present in gD-2.     

Lymphocyte zinc metabolism in disease

Lymphocytes were obtained from two patients with paroxysmal nocturnal hemoglobinuria as well as from apparently healthy controls and from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia. Subsequently, several aspects of zinc metabolism were studied in vitro in short-term cultures of these lymphocytes in order to assess lymphocyte functional capacity. The results of mitogen stimulation and zinc uptake studies for lymphocytes from donors with paroxysmal nocturnal hemoglobinuria were similar to those obtained for leukemic lymphocytes. The results of studies to determine the inducibility of the low molecular weight zinc-binding protein, metallothionein, by zinc were complicated by the decrease in overall protein synthesis in lymphocytes from donors in the paroxysmal nocturnal hemoglobinuria. It is proposed that paroxysmal nocturnal hemoglobinuria is indeed a clonal disorder and the relationship between lymphocytes in this disorder and leukemic lymphocytes is discussed.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

Human monocytes selectively bind to cells expressing the tumorigenic phenotype

Summary The characteristics of the binding of human monocytes to tumor cells were studied by a newly developed microassay. First, we determined the kinetics and optimal conditions of the binding. Monocytes recognized and bound to tumor cells very rapidly within 10–20 min of cellular interaction. Binding was also more efficient at 37°C suggesting that active metabolism of monocytes is required. Second, we determined that selective binding of monocytes to cells with tumorigenic phenotypes occurs. For this purpose, lymphocytic leukemia cell lines versus normal lymphocytes, and tumorigenic versus nontumorigenic hybrids from the same parental lines were compared as the targets of the binding assay. In both cases, neoplastic cells were selectively bound by monocytes. Although tumor cells were bound rapidly and selectively by monocytes, initial recognition and binding did not necessarily lead to subsequent tumor cell lysis. This is based on the observation that some tumorigenic parental and hybrid lines were avidly bound by monocytes yet not subsequently killed in a cytotoxicity assay.This work was supported in part by a grant from the National Institutes of Health CA42992 and a grant from the Kleberg foundation Abbreviations used: [¹²⁵I]IdUrd [¹²⁵I]iododeoxyuridine; rIFN-, recombinant human interferon ; IL-1, interleukin 1; rTNF, recombinant human tumor necrosis factor     

Complotypes in individuals of African origin: frequencies and possible extended MHC haplotypes

We analyzed the frequency distribution of 106 complotypes [four allele sets of the major histocompatibility complex (MHC) genes for the complement proteins factor B, C2, C4A, and C4B] from 32 Black families residing in Boston and Washington, DC. Twenty-five different complotypes were identified, among which there were four complotypes that had not been previously observed in our large database of complotypes compiled from family studies of Boston Caucasians and that are, presumably, unique to individuals of African origin. These four African-derived complotypes areFC(1,90)0, FC63, S1C2,17, andSC(3,2,90)0. The frequencies of two of these four unique Black complotypes,FC(1,90)0 andFC63, were increased significantly when compared to Caucasians (p_corr <0.00042, p_corr=0.00294, respectively). The complotypeFC(1,90)0 was in positive linkage disequilibrium withHLA-DR3 haplotypes containing theB locus antigens Bw42, Bw52, Bw53, and Bw58, whileFC63 was associated withHLA-Bw70,-DR5. These findings demonstrate the extensive polymorphism of complotypes in Blacks, and also suggest that it may be possible to define unique extended haplotypes of African origin.     

Molecular genetic study of human arginase deficiency

We have explored the molecular pathology in 28 individuals homozygous or heterozygous for liver arginase deficiency (hyperargininemia) by a combination of Southern analysis, western blotting, DNA sequencing, and PCR. This cohort represents the majority of arginase-deficient individuals worldwide. Only 2 of 15 homozygous patients on whom red blood cells were available had antigenically cross-reacting material as ascertained by western blot analysis using anti-liver arginase antibody. Southern blots of patient genomic DNAs, cut with a variety of restriction enzymes and probed with a near-full-length (1,450-bp) human liver arginase cDNA clone, detected no gross gene deletions. Loss of a TaqI cleavage site was identified in three individuals: in a homozygous state in a Saudi Arabian patient at one site, at a different site in homozygosity in a German patient, and in heterozygosity in a patient from Australia. The changes in the latter two were localized to exon 8, through amplification of this region by PCR and electrophoretic analysis of the amplified fragment after treatment with TaqI; the precise base changes (Arg291X and Thr290Ser) were confirmed by sequencing. It is interesting that the latter nucleotide variant (Thr290Ser) was found to lie adjacent to the TaqI site rather than within it, though whether such a conservative amino acid substitution represents a true pathologic mutation remains to be determined. We conclude that arginase deficiency, though rare, is a heterogeneous disorder at the genotypic level, generally encompassing a variety of point mutations rather than substantial structural gene deletions.     

Embodying illness,embodying cancer

Individuals and societies embody illnesses in different ways, in part determined by the way a person knows and lives his or her diagnosis and prognosis. Based on research in Northern Italy, on the experiences and meanings of cancer and on the practice of nondisclosure of the diagnosis, we find nondisclosure reflects a world divided - life/death, good/bad, mind/body — with the unwanted converted to other. The strong association of cancer with death, suffering, and hopelessness in much of Italy, coupled with the tremendous power attributed to naming and sentencing makes nondisclosure a major mechanism for keeping the condemned in this social world, and keeping death, decay, and suffering in the other. It is the social reality that is dominant here, such that informing a patient of cancer can be tantamount to social death.

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Résumé Les individus et les sociétés incorporent la maladie de façon différente, déterminée en partie de comment une personne connait et vit son diagnostic et prognostic. A partir de la recherche des experiences des significations du cancer et de la pratique de ne pas dire la diagnostic au Nord de l'Italie, on a remarqué que l'habitude de ne rien dire reflète un monde séparé entre la vie et la mort, entre le bon et le mal, entre l'esprit et le corps, de sorte que ce qui West pas voulu soit transfomé en l'autre. L'association forte du cancer à la mort, à la souffrance, au désespérance en toute l'Italie, unie au grand pouvoir donne au fait de dénommer et de donner une sentence, rend ne pas dire un méchanisme important pour garder le condamnd dans ce monde social et pour garder la mort, la décadence et la souffrance dans l'autre. C'est la réalité sociale qui est ici dominante, tel que le fait d'informer un patient de cancer soit comme une mort sociale.