Association of mouse fibrinogen-like protein with murine hepatitis virus-induced prothrombinase activity.

Previously, we demonstrated induction of a unique macrophage prothrombinase during infection of BALB/cJ mice by mouse hepatitis virus strain 3 (MHV-3). By immunologic screening, a clone representing this prothrombinase was isolated from a cDNA library and sequenced. The sequence identified this clone as representing part of a gene, musfiblp, that encodes a fibrinogen-like protein. Six additional clones were isolated, and one clone, p11-3-1, encompassed the entire coding region of musfiblp. Murine macrophages did not constitutively express musfiblp but, when infected with MHV-3, synthesized musfiblp-specific mRNA. musfiblp mRNA induction was earlier and significantly greater in BALB/cJ than A/J macrophages. Prothrombinase activity was demonstrated when musfiblp was expressed from p11-3-1 in RAW 264.7 cells. These data suggest that musfiblp encodes the MHV-induced prothrombinase.     

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.     

Tunicamycin-mediated depletion of insulin receptors in 3T3-L1 adipocytes.

Tunicamycin, an antibiotic that inhibits protein glycosylation, elicited a rapid depletion of insulin binding activity at the surface of 3T3-L1 adipocytes. Disappearance of insulin receptors occurred more rapidly in the presence of tunicamycin than when protein synthesis was inhibited by cycloheximide and was accompanied by a diminution in sensitivity of the adipocytes to the acute effects of insulin and anti-insulin receptor antibody on hexose uptake and metabolism.     

Bacterial organisms suitable for filamentous cell immobilization

Summary Several bacterial strains commonly used in biotechnology have been studied for induced filamentous growth at a wide range of sublethal 18-crown-6 concentrations. Only the rod shape bacteriaEscheriehia coli, Salmonella typhimurium andBacillus sp exhibit filamentous morphology, irrespective of whether the organism is gram stain positive or negative.     

Effect of temperature on the biaxial mechanics of excised lung parenchyma of the dog.

The influence of temperature on the mechanical properties of excised saline-filled lung parenchyma of the dog was studied at low lung volume. The motivation of this study was to determine whether lung tissue material without the influence of surface tension undergoes a phase transition in the 20-40 degrees C range, as does synthetic elastin studied by Urry in 1984-1986. Dynamic biaxial and uniaxial tensile tests were done, and strain vs. Lagrangian stress curves were recorded during slow cooling and heating between 40 and 10 degrees C. To emphasize the effects of elastin, strains (defined as stretch ratio minus one) were kept below 30%. A slight decrease in compliance occurred with cooling over the entire temperature range. This effect may be attributed to collagen. It was accompanied by a gradual increase in length as the tissue cooled, an effect that may be attributed to elastin. This process was partially reversible with reheating. However, this effect is in contrast with the sudden drastic change in mechanical properties of synthetic elastin described by Urry. Hysteresis, creep, and stress relaxation were small at these low strains. Possible causes of these effects are discussed.     

Interleukin 2-induced tyrosine phosphorylation. Interleukin 2 receptor beta is tyrosine phosphorylated

Interaction of interleukin 2 (IL2) with its high affinity membrane receptor complex (IL2R) is sufficient to induce proliferation of T lymphocytes. However, the biochemical mechanisms by which IL2 induces this process remain unresolved. The IL2R complex consists of at least two distinct polypeptides that bind IL2, a 75-kDa intermediate affinity subunit (IL2R beta) and a 55-kDa low affinity subunit (IL2R alpha). As indicated by Western blotting with anti-phosphotyrosine-specific antibodies and confirmed by phosphoamino acid analysis, we now demonstrate that interaction of the T cell growth factor interleukin 2 (IL2) with its high affinity receptor on IL2-sensitive human peripheral blood lymphoblasts induces tyrosine phosphorylation of proteins of 92, 80, 78, 70-75, and 57 kDa. IL2 induced tyrosine phosphorylation in YT 2C2 cells which express only the 75-kDa intermediate affinity IL2 binding molecule (IL2R beta) but not in cells which either express only the 55-kDa low affinity IL2 receptor molecule (IL2R alpha) or no IL2-binding sites. Therefore, IL2R beta, in the absence of IL2R alpha, appears sufficient to transduce the transmembrane signal leading to tyrosine phosphorylation. Two different antibodies reactive with phosphotyrosine specifically immunoprecipitated IL2R beta cross-linked to radiolabeled IL2. These findings suggest that IL2R beta is a substrate for the tyrosine kinase which is activated by IL2 binding to its receptor. Thus, like several other growth factor receptors, activation of the IL2R results in an increase in tyrosine phosphorylation with the receptor itself serving as one substrate.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.