Synthesis of alpha subunit of human chorionic gonadotrophin by presumptive HeLa cells

Summary Several cell lines, originally thought to be derived from a human placenta at term but possibly HeLa-contaminated, have been studied. These cells secrete a protein indistinguishable immunochemically from the alpha subunit of chorionic gonadotropin but not the beta subunit of chorionic gonadotropin or placental lactogen. Complete chorionic gonadotropin was detected but amounted to less than 1% of the level of the alpha subunit. The cells also produce an alkaline phosphatase similar to placental alkaline phosphatase in immunochemical, gel-electrophoretic, and heat-denaturation properties. They induce tumor growth when inoculated into nude mice. These cells are aneuploid and have a model chromosome number of 66. The common HeLa karyologic markers, designated 1, 2, and 3, and A-type glucose-6-phosphate dehydrogenase are present in these cells. HeLa cells have not previously been shown to secrete theα subunit of hCG.     

Radioimmunoassay of prostaglandins E1, E2, and F2α in unextracted plasma, serum and myocardium

A radioimmunoassay procedure for the determination of PGE₁, PGE₂, and PGF₂α is presented. The procedure involves the pre-precipitation of each prostaglandin specific antiserum with the precipitating antisera (ARGG), and the use of these antisera mixtures in assaying for PGE₁, PGE₂, and PGF₂α. Applicability of the methods to unextracted plasma, serum and myocardial homogenate has been demonstrated through tests of specificity, recovery, reproducability and parallelism. A mathematical correction for cross-reactivity between PGE₁ and PGE₂, and their opposing antisera is given. To demonstrate the utility of the methodology in differentiation of experimental variables, prostaglandin concentrations in unincubated serum, incubated serum, and the rate of prostaglandin production in serum of dogs are given.     

Amino acid sequence and immunological properties of chachalaca egg white lysozyme

Summary The amino acid sequence of lysozyme c from chachalaca egg white was determined. Like other bird lysozymes c, that of the chachalaca has 129 amino acid residues. It differs from other avian lysozymes c by 27 to 31 amino acid substitutions as well as by being devoid of phenylalanine. It contains substitutions at 9 positions which are invariant in the other 7 bird lysozymes of known sequence. Although the chachalaca is classified zoologically in the order Galliformes, which includes chickens and other pheasant-like birds, its lysozyme differs more from those of pheasant-like birds than do the lysozymes c of ducks. Phylogenetic analysis of the sequence comparisons confirms that the lineage leading to chachalaca lysozyme c separated from that leading to other galliform lysozymes c before the duck lysozyme c lineage did. This indicates a contrast between protein evolution and evolution at the organismal level. Immunological comparison of chachalacalysozyme c with other lysozymes of known sequence provides further support for the proposal that immunological cross-reactivity is strongly dependent on degree of sequence resemblance among bird lysozymes.103rd communication on lysozymes from the Laboratory of P. Jollès. Supported in part by grants from C.N.R.S. (ER 102), I.N.S.E.R.M. (Groupe de recherche U-116), N.S.F. (GB-42028X), and N.I.H. (GM-21509).     

Condensin I and condensin II proteins form a LINE-1 dependent super condensin complex and cooperate to repress LINE-1

Condensin I and condensin II are multi-subunit complexes that are known for their individual roles in genome organization and preventing genomic instability. However, interactions between condensin I and condensin II subunits and cooperative roles for condensin I and condensin II, outside of their genome organizing functions, have not been reported. We previously discovered that condensin II cooperates with Gamma Interferon Activated Inhibitor of Translation (GAIT) proteins to associate with Long INterspersed Element-1 (LINE-1 or L1) RNA and repress L1 protein expression and the retrotransposition of engineered L1 retrotransposition in cultured human cells. Here, we report that the L1 3′UTR is required for condensin II and GAIT association with L1 RNA, and deletion of the L1 RNA 3′UTR results in increased L1 protein expression and retrotransposition. Interestingly, like condensin II, we report that condensin I also binds GAIT proteins, associates with the L1 RNA 3′UTR, and represses L1 retrotransposition. We provide evidence that the condensin I protein, NCAPD2, is required for condensin II and GAIT protein association with L1 RNA. Furthermore, condensin I and condensin II subunits interact to form a L1-dependent super condensin complex (SCC) which is located primarily within the cytoplasm of both transformed and primary epithelial cells. These data suggest that increases in L1 expression in epithelial cells promote cytoplasmic condensin protein associations that facilitate a feedback loop in which condensins may cooperate to mediate L1 repression.     

Identification of a novel adhesion molecule in human leukocytes by monoclonal antibody LB-2

Monoclonal antibody LB-2 to a surface antigen on human B cells, lymphoblast, monocytes and vascular endothelial cells largely inhibited adhesion among Epstein Barr virus-immortalized normal B cells (EBV-B) and concanavalin A-stimulated blood mononuclear cells (Con A-BMC) before and after phorbol ester treatment. The antibody inhibited to a lesser extent phorbol ester-induced aggregation of monocytes, U937 cells and fresh BMC and had virtually no inhibitory effect on the adhesion among enriched T cells and granulocytes. A surface glycoprotein band of 84 kDa was obtained from EBV-B cells by immunoprecipitation and gel electrophoresis. Immunological and biochemical studies clearly distinguished this molecule from gp90 and associated glycoproteins which also mediate leukocyte adhesion.     

Imipenem and expression of multidrug efflux pump in Enterobacter aerogenes

Imipenem is often used to treat intensive care unit patients infected by Enterobacter aerogenes, but it is leading to an increasing number of antibiotic resistant strains. Clinical isolates and imipenem resistant variants presented a high level of resistance to beta-lactam antibiotic group and to chemically unrelated drugs. We report here that imipenem selects strains which contain active efflux pumps ejecting various unrelated antibiotics including quinolones, tetracycline, and chloramphenicol. An increase of AcrA, an efflux pump component, was observed in the imipenem resistant variants. The overexpression of marA, involved in the genetic control of membrane permeability via porin and efflux pump expression, indicated the activation of the resistance genetic cascade in imipenem resistant variants.     

Uptake and subcellular partitioning of trivalent metals in a green alga: comparison between Al and Sc

Despite 40+ years of research on aluminum (Al) toxicity in aquatic organisms, Al transport mechanisms through biological membranes, and the intracellular fate of Al once assimilated, remain poorly understood. The trivalent metal scandium shares chemical similarities with Al and, unlike Al, it has a convenient radioactive tracer (Sc-46) allowing for relatively simple measurements at environmentally relevant concentrations. Thus, we investigated the potential of Sc to substitute for Al in uptake and intracellular fate studies with the green alga Chlamydomonas reinhardtii. Short-term (<60 min) competitive uptake experiments indicated that Al does not inhibit Sc influx, implying that these metals do not share a common transport mechanism. Also, internalized Al concentrations were ~4 times higher than Sc concentrations after long-term (72 h) exposures under similar conditions (4.5 μM Al_T or Sc_T, 380 μM F_T, pH 7.0, 3.8 pM Al _calc ³⁺ and 1.0 pM Sc _calc ³⁺ ). However, interesting similarities were observed in their relative subcellular distributions, suggesting possible common toxicity/tolerance mechanisms. Both metals mostly distributed to the organelles fraction and almost no association was found with the cytosolic proteins. The greatest difference was observed in the cellular debris fraction (membranes and nucleus) where Al was much more concentrated than Sc. However, it is not clear whether or not this fraction contained extracellular metal associated with the algal surface. To summarize, Sc does not seem to be an adequate substitute of Al for transport/uptake studies, but could be for investigations of toxicity/tolerance mechanisms in C. reinhardtii. Further work is needed to verify this latter suggestion.     

Further Daphniphyllum Alkaloids with Insecticidal Activity from the Bark of Daphniphyllum macropodum Miq.

Two new Daphniphyllum alkaloids, macropodumines J and K ( 1 and 2 , resp.), together with six known structurally related alkaloids, 3 – 8 , were isolated from the bark of Daphniphyllum macropodum Miq . The structures of the new compounds 1 and 2 were elucidated on the basis of a comprehensive analysis of their spectroscopic and chemical data. Macropodumine J ( 1 ) contains a CN group which is relatively rare in naturally occurring alkaloids. All isolated compounds were tested for their insecticidal activities against a number of insect species. Daphtenidine C ( 5 ) is the most active compound against Plutella xylostella. This is the first report of insecticidal properties of Daphniphyllum alkaloids.     

Analysis of gamma c-family cytokine target genes. Identification of dual-specificity phosphatase 5 (DUSP5) as a regulator of mitogen-activated protein kinase activity in interleukin-2 signaling

Interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21 form a family of cytokines based on their sharing the common cytokine receptor gamma chain, gamma(c), which is mutated in X-linked severe combined immunodeficiency (SCID). As a step toward further elucidating the mechanism of action of these cytokines in T-cell biology, we compared the gene expression profiles of IL-2, IL-4, IL-7, and IL-15 in T cells using cDNA microarrays. IL-2, IL-7, and IL-15 each induced a highly similar set of genes, whereas IL-4 induced distinct genes correlating with differential STAT protein activation by this cytokine. One gene induced by IL-2, IL-7, and IL-15 but not IL-4 was dual-specificity phosphatase 5 (DUSP5). In IL-2-dependent CTLL-2 cells, we show that IL-2-induced ERK-1/2 activity was inhibited by wild type DUSP5 but markedly increased by an inactive form of DUSP5, suggesting a negative feedback role for DUSP5 in IL-2 signaling. Our findings provide insights into the shared versus distinctive actions by different members of the gamma(c) family of cytokines. Moreover, we have identified a DUSP5-dependent negative regulatory pathway for MAPK activity in T cells.