Differentiation of the pulmonary surfactant system. Disaturated phosphatidylcholine accumulation in fetal rat lung in vivo and in vitro

Fetal rat lung was placed in organ culture at 15 days gestation (22 days total gestation period), before biochemical and morphological development of the pulmonary surfactant system. At the fifth day of culture numerous Type II cells containing lamellar bodies were present as determined by electron micrography. Phospholipid accumulation in the cultures increased abruptly beginning at 6 days in culture. The phospholipid which accumulated between the sixth and twelfth culture days was composed of 21--27% disaturated phosphatidylcholines. Both the percent of disaturated phosphatidylcholines in the phospholipid fraction and the qualitative pattern of accumulation as a function of time were similar to observations for fetal rat lung developing in vivo. The data presented provide evidence for development of the pulmonary surfactant system in organ culture in vitro.     

Successful vaccination strategies that protect aged mice from lethal challenge from influenza virus and heterologous severe acute respiratory syndrome coronavirus

Newly emerging viruses often circulate as a heterogeneous swarm in wild animal reservoirs prior to their emergence in humans, and their antigenic identities are often unknown until an outbreak situation. The newly emerging severe acute respiratory syndrome coronavirus (SARS-CoV) and reemerging influenza virus cause disproportionate disease in the aged, who are also notoriously difficult to successfully vaccinate, likely due to immunosenescence. To protect against future emerging strains, vaccine platforms should induce broad cross-reactive immunity that is sufficient to protect from homologous and heterologous challenge in all ages. From initial studies, we hypothesized that attenuated Venezuelan equine encephalitis virus (VEE) replicon particle (VRP) vaccine glycoproteins mediated vaccine failure in the aged. We then compared the efficacies of vaccines bearing attenuated (VRP(3014)) or wild-type VEE glycoproteins (VRP(3000)) in young and aged mice within novel models of severe SARS-CoV pathogenesis. Aged animals receiving VRP(3000)-based vaccines were protected from SARS-CoV disease, while animals receiving the VRP(3014)-based vaccines were not. The superior protection for the aged observed with VRP(3000)-based vaccines was confirmed in a lethal influenza virus challenge model. While the VRP(3000) vaccine's immune responses in the aged were sufficient to protect against lethal homologous and heterologous challenge, our data suggest that innate defects within the VRP(3014) platform mediate vaccine failure. Exploration into the mechanism(s) of successful vaccination in the immunosenescent should aid in the development of successful vaccine strategies for other viral diseases disproportionately affecting the elderly, like West Nile virus, influenza virus, norovirus, or other emerging viruses of the future.     

Amino-terminal sequence of a phospholipid transfer protein from rat lung

A phospholipid transfer protein from rat lung has been characterized in terms of the amino-terminal sequence. The sequence is Val-Leu-Leu-Lys-Glu-Tyr-Arg-Val-Ile-Leu-Pro-(Val)-His-Val-Asp-Glu-Tyr-Gln-Val- Gly. Comparison of the amino-terminal sequence of the protein from lung with sequences from phosphatidylcholine transfer protein and non-specific phospholipid transfer protein from bovine liver revealed no apparent sequence homology. The sequence showed no homology with fatty acid binding proteins or cellular retinoid binding proteins.     

Nitrate reductase from Chlorella vulgaris. Reaction with manganese (III) pyrophosphate and with ferric o-phenanthroline.

Millimolar concentrations of tervalent manganese pyrophosphate can partially activate nitrate reductase which has been inactivated with NADH and HCN. The tervalent manganese complex is nevertheless not reduced by NADH in the presence of the enzyme, that is, it is not a substrate for the diaphorase moiety of the nitrate reductase. Ferric o-phenanthroline, on the other hand, is a good diaphorase substrate, but fails to activate the inactive enzyme.     

The lung lamellar body as a functioning membrane in protein-catalyzed phosphatidylcholine transfer

Lung lamellar bodies and liver mitochondria were used to demonstrate that soluble phospholipid transfer proteins from lung transfer phosphatidylcholine to both of these acceptors. The initial rate of transfer to lung lamellar bodies is about half that of the rate of transfer to the liver mitochondria when both acceptor membranes are present at saturating concentrations. Phosphatidylcholine unilamellar vesicles were used to demonstrate that the fatty acyl composition of the membrane phosphatidylcholine is a significant determinant of the rate of phosphatidylcholine transfer catalyzed by these proteins. The lamellar bodies have a unique phosphatidylcholine composition, and these studies suggest that this is an important factor in determining the lower initial rate of transfer to lamellar bodies. The studies have also characterized two phospholipid transfer proteins in rat lung in terms of isoelectric point. Isoelectric points for the two proteins which transfer phosphatidylcholine were found to be 5.6 ± 0.08 and 6.2 ± 0.03.     

Significant non-S-phase DNA synthesis visualized by flow cytometry in activated and in malignant human lymphoid cells

The development of a monoclonal antibody to the deoxynucleoside bromodeoxyuridine (BrdU), combined with two parameter flow cytometry, has allowed us to examine large numbers of cells for non-S-phase DNA synthesis. Three human lymphoid cell populations were studied to determine the level of deoxynucleoside (dN) incorporation as a function of DNA content. In each population, non-S-phase DNA synthesis was observed. In a rapidly growing human T-lymphoblastoid cell line (CCRF-CEM), 53% of dN incorporation occurred in G0/G1 plus G2 + M. In chronic lymphocytic leukemia (CLL) cells stimulated with tetradecanoylphorbol acetate (TPA), 45% of the observed burst in thymidine incorporation was found to be localized to G0/G1 cells. Non-S-phase incorporation was not, however, limited to neoplastic cells. Normal human peripheral blood B cells treated with the Cowan strain of Staphylococcus aureus (CSA) undergo a transient burst in thymidine incorporation, but do not go on to divide in the absence of other stimuli. Flow-cytometric analysis showed that 80% of this CSA-stimulated dN incorporation was into G0/G1 cells. These data are consistent with a more dynamic state of DNA synthesis than usually envisioned. Furthermore, the data show that although thymidine incorporation levels are related to incorporation of dN into DNA, they can be unrelated to cell proliferation.