Monocyte antigen CD14 is a phospholipid anchored membrane protein

A cDNA clone encoding the human monocyte antigen CD14 was isolated by transient expression in COS cells of a cDNA library prepared from phorbol diester-treated HL60 cells. RNA blot analysis showed abundant expression of a single mRNA species in mature monocytes and an increased expression of the mRNA following induction of differentiation in leukemic cell lines. The DNA blot hybridization pattern was consistent with a single-copy gene. The predicted amino acid sequence lacks the characteristic transmembrane domain and stop transfer motif of conventionally anchored membrane proteins. COS cells transfected with the CD14 cDNA released virtually all CD14 protein in soluble form following treatment with glycosyl phosphatidylinositol-specific phospholipase C, and CD14 immunoreactivity was absent from the affected monocytes of a patient with paroxysmal nocturnal hemoglobinuria (PNH). The data show that, to the limit of experimental sensitivity, all monocyte CD14 is joined to the plasma membrane by a phosphatidylinositol phospholipid.     

Procoagulant activity of reversibly acylated human factor Xa

The plasma clotting factors used to treat hemophiliacs who have developed inhibitory antibodies have a shared history of limited clinical safety and utility. To improve on existing bypass factors, we have developed a reversibly acylated form of human plasma factor Xa capable of providing a time-dependent release of procoagulant activity. Factor Xa was treated with p-amidinophenyl p'-anisate to generate anisoyl Xa. The chemical modification of the protein involves acylation of the active site serine residue of factor Xa. Anisoyl Xa deacylated in a time, pH, and temperature-dependent manner. Active factor Xa generated on deacylation of anisoyl Xa exhibited amidolytic and prothrombinase complex activities in in vitro assays, the level being comparable to those of untreated factor Xa. When Anisoyl Xa was infused into rabbits, active factor Xa was generated on deacylation of the acylated enzyme, which shortened the activated partial thromboplastin time (APTT) in a dose-dependent manner. The duration of effect on rabbit APTT could be directly correlated to the level of human plasma factor Xa. Because anisoyl Xa bypasses the "tenase" complex that is compromised in hemophilia A and B and is unaffected by inhibitory antibodies, it has the potential to be used as an effective bypass therapy.     

Nitric oxide interactions with cobalamins: biochemical and functional consequences

Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.     

Abnormal pregnancy: early diagnosis by US and serum chorionic gonadotropin levels

Simultaneous sonography and quantitative serum human chorionic gonadotropin (HCG) levels from 126 women with threatened abortion were compared. Of 56 women with normal outcome, 39 (70%) had a gestation sac greater than or equal to 5 mm in mean sac diameter, and in each case the HCG level was 1,800 milli-international units (mIU/ml) or greater. The serum HCG levels strongly correlated with the gestation sac sizes to a mean sac diameter of 25 mm. Of 70 abnormal pregnancies, 31 demonstrated a gestation sac. Of these, 20 women (65%) had disproportionately low HCG levels relative to sac size, including 12 in whom the HCG level was less than 1,800 mIU/ml. One woman with an early molar pregnancy had a disproportionately elevated HCG level. Correlation of sonograms with a simultaneous measurement of serum HCG level is a useful method for evaluating threatened spontaneous abortion. A disproportionately low HCG level relative to gestation sac size is evidence for an abnormal pregnancy.     

Anti-IgM induces transforming growth factor-beta sensitivity in a human B-lymphoma cell line: inhibition of growth is associated with a downregulation of mutant p53

We wished to examine the role of transforming growth factor-beta (TGF- beta) in the regulation of human lymphoma cell growth. The RL cell line is an immunoglobulin M (IgM)+, IgD+ B lymphoma cell line, which does not constitutively express receptors for TGF-beta, and thus has lost the ability to respond to the inhibitory effects of TGF-beta. We demonstrate here that anti-Ig antibodies can efficiently upregulate the expression of TGF-beta receptors and promote sensitivity to growth inhibition by TGF-beta. Furthermore, because TGF-beta has been shown to function in late G1 of the cell cycle, we examined the ability of TGF- beta to modulate two tumor suppressor proteins known to be critical regulators of the G1/S transition, Rb and p53. Rb is a 105- to 110-kD phosphoprotein, which has been shown to maintain its growth suppressive function when it is found in the hypophosphorylated state. Wild-type p53 is a 53-kD phosphoprotein that appears to be important in preventing cell-cycle progression and promoting apoptosis in cells with DNA damage, whereas mutant p53 can overcome those functions. We show here that TGF-beta treatment of phorbol myristate acetate (PMA) or anti- Ig-activated RL cells results in growth inhibition through a dual effect on Rb and mutant p53. After TGF-beta treatment, we observe a predominance of Rb in the hypophosphorylated, growth suppressive form. In addition, we show a decrease in levels of mRNA and protein for mutant p53. We also show that, although these changes are sufficient to halt progression through the cell cycle, the cells do not appear to undergo extensive programmed cell death following 72 hours of TGF-beta treatment. Thus, although these lymphoma cells maintain the capacity to be negatively growth regulated by TGF-beta, the ability of TGF-beta to induce apoptosis must be independently controlled.