Role of receptor-binding activity of the viral hemagglutinin molecule in the presentation of influenza virus antigens to helper T cells.

The concentration of antigen required to stimulate influenza virus-specific helper T cells was observed to be dependent upon the antigenic form bearing the relevant determinant: intact, nonreplicative virus was needed only in picomolar amounts, while denatured proteins, protein fragments, or synthetic peptides were required in micromolar concentrations for a threshold level of stimulation. Antigenic efficiency of intact virus was found to result from the attachment of virus to sialic acid residues on the surface of the antigen-presenting cell since spikeless viral particles lacking the hemagglutinin molecule were much less efficient antigens for helper T cells and continuous presence of hemagglutination-inhibiting antihemagglutinin antibodies reduced efficiency of stimulation by intact virus approximately 100-fold for both hemagglutinin and internal virion proteins. Influenza virus associated rapidly with antigen-presenting cells; less than 10 min at 20 degrees C was sufficient to introduce virus for a maximal level of T-cell stimulation. This rapid attachment was blocked by antibodies to the hemagglutinin or by pretreatment of the antigen-presenting cells with neuraminidase to remove the cellular virus receptor. Following viral adsorption by antigen-presenting cells, a lag period of 30 min at 37 degrees C was required for the expression of helper T-cell determinants. One early event identified was the movement of the virus to a neuraminidase-insensitive compartment, which can occur at 10 degrees C, but which was not equivalent to expression of helper T-cell determinants. Preincubation of cells with virus at 10 degrees C for 4 h reduced the lag period of helper T-cell determinant expression to 15 min when these cells were shifted to 37 degrees C, suggesting that transition of the virus to a neuraminidase-resistant state is a required step in presentation of T-cell antigenic determinants.     

The NK-1.1(-) mouse: a model to study differentiation of murine NK cells

The NK-1.1(-) mouse was constructed by weekly injections of monoclonal anti-NK-1.1 antibody from birth through adulthood. Spleen cells from these mice have decreased NK-1.1+ cells and null (Thy-1- and B220-) cells. Their splenic NK activity to YAC targets was low and was not enhanced by IFN-alpha or IFN-beta. Bone marrow (BM) of these NK-1.1(-) mice have normal precursors to NK cells: 1) NK activity could be generated from NK-1.1(-) BM cells cultured in rIL 2 for 5 to 6 days. These cultured BM cells expressed Qa-5, Thy-1, AsGm-1, and NK-1.1 antigens. The precursor cells of these BM cytotoxic cells are NK-1.1-; 2) transfer of BM cells from the NK-1.1(-) mice reconstituted the NK activity of irradiated, NK-depleted recipients. Lymphokine-activated killer cells could also be generated from spleens of these NK-1.1(-) mice. Therefore, the NK-1.1(-) mice were specifically depleted of mature cytotoxic NK cells, but not the NK-1.1- precursors of NK cells. This mouse model is valuable to study ontogeny and physiologic relevance of NK cells.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with α-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl-4-hydroxyquinoneN-oxide. CuSO₄ inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates → cytochromes 548,552 → cytochromes 554,557 → trimethylamine-N-oxide reductase → TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and / or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Growth of normal human mammary cells in culture

Summary Reduction mammoplasty tissue was used to obtain short-term cultures of human epithelial cell populations. Digestion of tissue with collagenase and hyaluronidase resulted in cell clusters (organoids) resembling ductal and alveolar structures; these could be separated by filtration from the stromal components. Epithelial outgrowth from these organoids was greatly enhanced by the addition of conditioned medium from other human epithelial and myoepithelial cell lines. Additionally, the mammary epithelial growth was stimulated by insulin, hydrocortisone, epidermal growth factor, and steroid hormones. With this enriched nutritional environment, active cell division could be maintained for 1 to 3 months and cells could be serially subcultured 1 to 4 times. This research was supported by Grant PDT-72 from the American Cancer Society and Grant CP-70510 from the National Institutes of Health.     

Clinical evaluation of an air embolism detection device

The clinical evaluation of the Sarns Air-Bubble Detector System in over 4,000 assorted operative procedures using cardiopulmonary bypass is described in this report. The system is designed to produce an alarm when a bolus of air enters the circuit. False alarms caused by electrical static within the operating room can be eliminated with a filter incorporated into the electrical circuit.     

Gibberellin-induced reorganization of spatial relationships of emerging leaf primordia at the shoot apical meristem in Hedera helix L.

The transition from spiral to distichous leaf arrangement during gibberellic-acid (GA₃)-induced rejuvenation in Hedera was studied in detail by scanning electron microscopy of the shoot apical meristem. The transition, which involves the initiation of about 14 new leaf primordia, is accomplished by progressive increments in the divergence angle between the leaf primordia from an initial average value of 138.9 ° until it approaches 180 °. This process is preceded, as well as accompanied, by an increased radial displacement of young leaf primordia away from the apical meristem. Although the width of the leaf primordia also increases, this is unlikely to be a causal factor since it occurs only late in the transition. The size of the primordium-free area of the apical meristem is also unlikely to be involved. Quantitative analysis shows that the divergence angle of consecutive leaf primordia commonly fluctuates between relatively large and small values. Thus the transitional stages form a spirodistichous arrangement in which the divergence angle within each pair of leaves is large relative to that between leaf pairs. The stimulation of the radial displacement of the leaf primordia and the associated phyllotactic transition may involve GA₃-induced modification in the spatial organization of cortical microtubules in the apical meristem and related changes in directional cell expansion.Abbreviations DA divergence angle - GA₃ gibberellic acid We thank Mr. Gilbert Ahlstrand for his advice regarding scanning electron microscopy. This paper is contribution of the University of Minnesota Agricultural Experimental Station No. 18,726.     

Class II MHC-restricted T cell determinants processed from either endosomes or the cytosol show similar requirements for host protein transport but different kinetics of presentation.

We have studied the role of APC protein transport in presentation of class II MHC-restricted T cell determinants of influenza virus glycoproteins that have distinct Ag processing requirements. Two I-Ed-restricted epitopes were analyzed: hemagglutinin (HA) 111-119, which is processed by the exogenous/endocytic pathway, and neuraminidase (NA) 79-93, which has a requirement for cytosolic processing. NA 79-93 is presented from infectious but not non-replicative virus under ordinary conditions. This requirement for viral biosynthesis could be bypassed by using a soluble inhibitor of NA,2,3-dehydro-2-deoxy-N-acetyl neuraminic acid (DDAN), to facilitate cytosolic introduction of virus. APC exposed to UV virus/DDAN present HA and NA determinants derived directly from proteins of the input virus particles. This allows presentation of both endocytically and cytosolically processed epitopes in the same experiment using noninfectious virus. The inhibitor brefeldin A (BFA) was used to interrupt host protein transport at various times relative to virus/DDAN addition. We observed that BFA added simultaneously with virus blocked recognition of NA 79-93 but not HA 111-119. This distinction was found to be based upon different expression kinetics of the HA and NA determinants. Expression of NA 79-93 required 6 to 9 h, whereas HA 111-119 was presented by 1 h after Ag addition. When APC were incubated with BFA at intervals before virus addition, presentation of HA 111-119 was also blocked as a function of time. Data indicate that about 5 h of BFA treatment is needed to deplete host protein pools required for presentation of I-Ed-restricted T cell determinants processed from either endosomes or the cytosol.     

Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase

A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.     

Properties of spores of Bacillus subtilis strains which lack the major small, acid-soluble protein.

Bacillus subtilis strains containing a deletion in the gene coding for the major small, acid-soluble, spore protein (SASP-gamma) grew and sporulated, and their spores initiated germination normally, but outgrowth of SASP-gamma- spores was significantly slower than that of wild-type spores. The absence of SASP-gamma had no effect on spore protoplast density or spore resistance to heat or radiation. Consequently, SASP-gamma has a different function in spores than do the other major small, acid-soluble proteins.