Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate.

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.     

Heparin/heparan sulfate is involved in attachment and spreading of mouse embryos in vitro

The involvement of embryonic cell surface proteoglycans in the attachment and outgrowth of cultured mouse embryos has been investigated. Several lines of evidence indicate that periimplantation stage blastocysts express heparin/heparan sulfate proteoglycans on their cell surfaces that can mediate embryo attachment and trophoblast outgrowth on a variety of matrices. First, in the presence of soluble heparin, the rate at which embryos attach and outgrow on laminin, fibronectin, or monolayers of uterine epithelial cells is reduced considerably. In the case of fibronectin, the rate of outgrowth in the presence of the heparin is slower than in the presence of the Arg-Gly-Asp-Ser-containing peptide that is recognized by a fibronectin receptor. Embryos also attach and exhibit a limited ability to outgrow on platelet factor IV, a heparin binding protein that does not possess the additional binding domains of laminin or fibronectin. Attachment on platelet factor IV is inhibited by heparin. Second, cell surface digestion of attachment-component embryos with heparinase, but not chondroitinase ABC, slows the rate of outgrowth on tissue culture plates in the presence of serum. Third, selective staining for sulfated molecules on the trophectoderm surface of periimplantation stage embryos indicates that such molecules are abundant and uniformly distributed on these cell surfaces. Last, heparin/heparan sulfate proteoglycans are detected as major cell surface components of embryos using vectorial labeling with lactoperoxidase and Na125I. Collectively, these data indicate that heparin/heparan sulfate-bearing molecules have a direct role in attachment and outgrowth of implantation stage blastocysts.     

A Pseudomonas stutzeri outer membrane protein inserts copper into N2O reductase

Among a set of frameshift mutagen (ICR-191; Polysciences, Inc.)-induced mutations that confer inability to grow anaerobically with N2O as the sole electron acceptor, one class was found that produced an inactive N2O reductase which lacked copper. All of these mutant strains failed to produce a 61,000-Mr protein located in the outer membrane. This protein, termed NosA, seems not to be responsible for bringing copper into the cell because the mutant strains and their parent were similarly sensitive to the copper content of the growth medium and no intermediate copper concentration in the medium permitted the mutant strains (nosA) to grow anaerobically with N2O as the sole electron acceptor. We conclude that NosA is necessary to insert copper into N2O reductase or to maintain it there.     

Photosensitized cleavage of dynein heavy chains. Cleavage at the V2 site by irradiation at 365 NM in the presence of oligovanadate

Irradiation of the outer-arm dynein ATPase from sea urchin sperm flagella at 365 nm in the presence of 50-200 microM vanadate (Vi) and 1 mM manganese acetate, in the absence of ATP, cleaves the alpha and beta heavy chains at a specific site, termed the V2 site, to form discrete peptides of Mr approximately 260,000 and 170,000 from the alpha chain and of Mr approximately 255,000 and 175,000 from the beta chain, with a yield of 80%. This cleavage at the V2 site is not correlated with any direct effect on the dynein ATPase activity. In the presence of 100 microM Vi, the half-times for cleavage of the alpha and beta chains are about 12 and 50 min, respectively. The rate of heavy chain cleavage shows a sigmoidal dependence upon Vi concentration, with half-maximal rate occurring at 58 +/- 7 microM, consistent with the chromophore responsible for cleavage being tri-vanadate. Addition of 10 microM ATP or ADP, or of 100 microM CTP or UTP, to the irradiation medium inhibits cleavage at the V2 site, and results in a slow cleavage occurring at the V1 site described previously. The peptides produced by sequential cleavage at the V2 and then the V1 sites indicate that the sites are separated by about 100,000 Da along the length of each heavy chain. Photoaffinity labeling with [alpha-32P] 8-azidoadenosine 5'-triphosphate (8-N3ATP) gives specific incorporation of 32P into both the Mr 255,000 and 175,000 peptides of the beta chain but into only the Mr 260,000 peptide of the alpha chain. These results suggest that V2 cleavage occurs on a loop of the heavy chain that forms part of the ATP-binding site, close to the locus of 8-N3ATP attachment.     

Identification of cellular mechanisms operational in vivo during the regression of established pulmonary metastases by the systemic administration of high-dose recombinant interleukin 2

The systemic administration of high-dose recombinant IL 2 mediated significant reductions of established 3-day pulmonary micrometastases from both weakly immunogenic and nonimmunogenic sarcomas. However, when treatment with IL 2 was delayed for 10 days after the injection of tumor cells in an attempt to treat grossly visible pulmonary macrometastases, only those established from weakly immunogenic sarcomas remained susceptible. Established 10-day pulmonary nodules from the nonimmunogenic sarcomas became refractory to IL 2 therapy. We utilized selective depletion of lymphocyte subsets in vivo by the systemic administration of specific monoclonal antibodies to cells bearing either the L3T4 or Lyt-2 marker or a heteroantiserum to cells bearing the ASGM-1 glycosphingolipid to identify lymphocytes involved in IL 2-induced tumor regression. Cells with potent lymphokine-activated killer (LAK) activity against fresh tumor targets in vitro were identified in the lungs of IL 2-treated mice. By flow cytometry analysis, the majority of these effector cells were Thy-1+, L3T4-, Lyt-2-, ASGM-1+. Depletion in vivo of ASGM-1+ cells before the onset of IL 2 administration eliminated the successful therapy of 3-day pulmonary metastases from nonimmunogenic sarcomas, with concurrent elimination of LAK cell activity in the lungs. In mice with 3-day pulmonary metastases from weakly immunogenic sarcomas, both Lyt-2+ cells and ASGM-1+ cells were involved in IL 2-mediated tumor regression, but Lyt-2+ cells appeared to be the more potent mediator in the response. Lyt-2+ cells were also involved in the elimination of grossly visible 10-day macrometastases from these weakly immunogenic tumors. Depletion of L3T4+ cells had no effect on tumor regression. Thus, although LAK effectors derived from ASGM-1+ precursors can eliminate pulmonary micrometastases regardless of tumor immunogenicity, Lyt-2+ cells are predominant effectors in the elimination of both pulmonary micro- and macrometastases from weakly immunogenic sarcomas.     

矮生型苹果苗的预选标志

用淀粉凝胶电泳,分析苹果实生苗新梢皮层的过氧化物酶同工酶,其酶9带(A_9)与苹果的矮生习性相关,且不受树龄、苹果类型以及栽培条件等影响。在矮生型苹果育种中,A_9可以作为预选标志,其准确率为86.14%。     

Estrogen influences dolichyl phosphate distribution among glycolipid pools in mouse uteri

The steroid hormone 17 beta-estradiol dramatically induces uterine N-linked glycoprotein assembly [Dutt, A., Tang, J.-P., Welply, J. K., & Carson, D. D. (1986) Endocrinology (Baltimore) 118, 661-673]. To determine the role that dolichyl phosphate availability plays in this induction, we studied the effects of estrogen priming on the content of dolichyl phosphate and the distribution of dolichyl phosphate among various glycolipids in uteri. Dolichol-linked saccharides were metabolically labeled to equilibrium with either [3H]glucosamine or [3H]mannose and extracted from primary explants of uterine tissue. The amount of dolichol-linked saccharide was calculated from the specific radioactivity determined for the corresponding sugar nucleotides extracted from the tissues. The major dolichol-linked saccharides identified were mannosylphosphoryldolichol (MPD), oligosaccharylpyrophosphoryldolichol (OSL), and N,N'-diacetylchitobiosylpyrophosphoryldolichol (CBL). Estrogen increased the levels of MPD and OSL 4-fold; however, CBL levels did not change. After 3 days of treatment, the levels of these glycolipids were very similar to those in uteri from pregnant mice. Remarkably, MPD constituted 90-95% of dolichol-linked saccharides detected under all conditions. The tissue contents of total dolichyl phosphate and alkali-labile dolichyl phosphate, presumably MPD, were estimated by liquid chromatography. The levels of alkali-labile dolichyl phosphate determined in this way were in good agreement with the values estimated for MPD by metabolic labeling; moreover, alkali-labile dolichyl phosphate constituted 50-98% of the total dolichyl phosphate pool. The variations in MPD content depended upon the steroid hormone influence, most notably that of estrogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Flow profiles and wall shear stress distribution at a hemodialysis venous anastomosis: preliminary study

The phasic velocity field in the vicinity of the venous anastomosis in a hemodialysis angioaccess arteriovenous fistula loop graft (AVLG) is investigated employing a laser Doppler anemometer (LDA) system. Detailed LDA velocity profiles are obtained by sectional survey performed in a transparent, elastic flow model which was fabricated to represent the geometry of the AVLG system under physiological pressure and flow waveforms. The geometry of the flow model was based on a silicone rubber cast obtained from an experimental dog model. In the present study, detailed distribution of velocity profiles is obtained. The distribution of wall shear stress in the model is computed from the slope of the local velocity profiles near the wall. The relationship between the results obtained by flow visualization and the LDA measurement is discussed.     

Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system.

We developed highly sensitive shuttle vector systems for detection of mutations formed in human cells using autonomously replicating derivatives of Epstein-Barr virus (EBV). EBV vectors carrying the bacterial lacI gene as the target for mutation were established in human cells and later returned to Escherichia coli for rapid detection and analysis of lacI mutations. The majority of the clonal cell lines created by establishment of the lacI-EBV vector show spontaneous LacI- frequencies of less than 10(-5) and are suitable for studies of induced mutation. The ability to isolate clonal lines represents a major advantage of the EBV vectors over transiently replicating shuttle vectors (such as those derived from simian virus 40) for the study of mutation. The DNA sequence changes were determined for 61 lacI mutations induced by exposure of one of the cell lines to N-nitroso-N-methylurea. A total of 33 of 34 lacI nonsense mutations and 26 of 27 missense mutations involve G X C to A X T transitions. These data provide support for the mutational theory of cancer.