Fetal lung surfactant lipid synthesis from glycogen during organ culture

The role of fetal lung glycogen as a precursor for lipids during late gestational development was explored by a combination of in vivo labeling with [U-14C]glucose, administered directly to rat fetuses at 18.5 days, and in vitro assessment using an organ explant culture system. Our major objectives were to demonstrate that radioactivity was transferred specifically and preferentially to surfactant lipids, as glycogenolysis occurred, and to determine the molecular distribution of 14C labeling in newly synthesized phosphatidylcholine (PC). Surfactant and residual (non-surfactant) lipids were separated by sucrose density gradient centrifugation, and other subcellular fractions such as microsomes were isolated by subsequent centrifugations. After 72 h of culture, there was a 5.7-fold increase in the concentration of PC in the surfactant fraction, which contributed 8.8% of total PC at the beginning and 29.6% (P less than 0.001) at the end of the 72 h period. The labeling of PC in the surfactant fraction increased markedly during culture, but there was no significant change in the residual fraction or microsomal PC. Hydrolysis of surfactant PC indicated that the radioactivity was predominantly located in the fatty acyl portion of the molecule, both before and after culture; however, PC glycerol labeling also increased significantly during culture. The distribution of PC radioactivity was similar in the residual fraction and microsomes, with the majority of 14C in the fatty acids. Neutral lipid radioactivity also increased significantly in both the surfactant (240%) and residual (136%) fractions. Quantitation of the changes in radioactivity among subcellular components during lung explant culture indicated that the greatest decrease occurred in glycogen, whereas only lipids, particularly those of the surfactant fraction, were found to show significant increases. These results support the hypothesis that glycogen, which accumulates in fetal lung prior to augmented surfactant production, can supply precursors for synthesis of functionally essential pulmonary phospholipids.     

Optimization of fetal lung organ culture for surfractant biosynthesis

Summary Lung organ culture has been a widely used system for studying differentiation and maturation of alveolar epithelium through various culture conditions. The purpose of this work was to carefully characterize in vitro lung biochemical diffeentiation through isolation of surfactant fraction from tissue and to search for optimal culture conditions. Fetal rat lung was explanted on the 18th gestational day for studying glycogen storage, and on the 20th gestational day for studying surfactant accretion, and cultivated for 48 h. Morphologic differentiation was studies byelectron microscopy tissue explanted on the 17th or 18th gestational days and cultivated for various times. Glycogen storage was greater on fluid medium, although less than occurring in vivo. Cellular integrity and surfactant accumulation were maximal on a semisolid medium containing 0.5% agar. Use of O₂-CO₂ instead of air-CO₂ for gassing the explants slighlty decreased phospholipid accumulation. Among media used in previous lung culture studies, Waymouth MB 752/1 was the only one to allow net glycogen accumulation in vitro. The most favorable media for surfactant phospholipid accretion were Waymouth MB 752/1, Eagle’s minimum essential and its Dulbeccco’s modification, CMRL 1066, and NCTC 109. They allowed a 12- to 14-fold increase of surfactant fraction phospholipids in vitro, which is similar to the increase occurring in vivo during the same peiod. Ham’s F10 and F12 media allowed a six fold increase. RPMI 1640 and medium 199 (M199) allowed only a three fold increase. Phospholipid concentration in nonsurfactant fraction only doubled during culture, and differences between various media were much less marked. DNA concentration changed little during culture. Morphologic differentiation of epithelial cells was advanced as compared with in vivo timing in a medium allowing maximal surfactant accretion (Waymouth MB 752/1) but not in a medium allowing low surfactant increase (RPMI 1640). The possible role of compositional differences between media is discussed.     

Maturation of a 100 kDa protein associated with preribosomes in Chinese hamster ovary cells

A 100 kDa nucleolar protein which is transitorely associated with preribosomes in the nucleoli of Chinese hamster ovary cells has been found to be specifically cleaved by a thiol protease. During an in vitro incubation of nucleoli, the 100 kDa protein is processed into eight different proteins which are detected by immunoreaction with a serum raised against the 100 kDa protein. qualitative and quantitative variations in the maturation products of the 100 kDa protein are obtained by in vitro incubation of the 60S and 80S preribosomes. The 100 kDa protein has been purified to homogeneity with the protease activity still associated. The properties of the enzyme are described and its role in the maturation of preribosomes is discussed.     

Urinary clearance of noradrenaline in the dog]

Glomerular filtration rate (GFR) and noradrenaline clearance are highly correlated in anesthetized dogs (ratio CNA-3H/GFR approximately 1).     

Knockout mice as model systems for studying nm23/NDP kinase gene functions. Application to the nm23-M1 gene

Mice carrying a homozygous germ-line mutation in the nm23-M1 gene that eliminates its protein expression and drives expression of -galactosidase by nm23-M1 promoter have been generated. nm23-M1 gene inactivation is not teratogenic and the pups can grow to adult age without apparent health problems. However, they undergo a growth retardation and knocked out females cannot feed their pups. Both effects are background dependent. -galactosidase mapping of nm23-M1 promoter activation during embryogenesis shows that the nm23-M1 gene is principally expressed in epithelial layer of tissues which require inductive epithelial–mesenchymal interactions for their formation. In conclusion, invalidated mice could be interesting models to analyze the role of nm23-M1 on signal transduction pathway regulation, or cancer induction and proliferation.     

Distinctive features of Drosophila alternative splicing factor RS domain: implication for specific phosphorylation, shuttling, and splicing activation

The human splicing factor 2, also called human alternative splicing factor (hASF), is the prototype of the highly conserved SR protein family involved in constitutive and regulated splicing of metazoan mRNA precursors. Here we report that the Drosophila homologue of hASF (dASF) lacks eight repeating arginine-serine dipeptides at its carboxyl-terminal region (RS domain), previously shown to be important for both localization and splicing activity of hASF. While this difference has no effect on dASF localization, it impedes its capacity to shuttle between the nucleus and cytoplasm and abolishes its phosphorylation by SR protein kinase 1 (SRPK1). dASF also has an altered splicing activity. While being competent for the regulation of 5' alternative splice site choice and activation of specific splicing enhancers, dASF fails to complement S100-cytoplasmic splicing-deficient extracts. Moreover, targeted overexpression of dASF in transgenic flies leads to higher deleterious developmental defects than hASF overexpression, supporting the notion that the distinctive structural features at the RS domain between the two proteins are likely to be functionally relevant in vivo.     

Inhibitory actions of ceramide upon PKC-epsilon/ERK interactions

We have previously shown that interleukin-1receptor-generated ceramide induces growth arrest in smooth musclepericytes by inhibiting an upstream kinase in the extracellularsignal-regulated kinase (ERK) cascade. Here, we now report themechanism by which ceramide inhibits ERK activity. Ceramide renders thehuman embryonic kidney 293 cells (HEK 293) resistant to the mitogenicactions of growth factors and activators of protein kinase C (PKC). A role for PKC to mediate ceramide inhibition of growth factor-induced ERK activity and mitogenesis is suggested, as exogenous ceramide directly inhibits both immunoprecipitated and recombinant PKC- activities. To confirm that PKC- is necessary for ceramide-inhibited ERK activity, HEK 293 cells were transfected with a dominant-negative mutant of PKC- (PKC-). These transfected cells respond toinsulin-like growth factor I (IGF-I) with a significantly decreased ERKactivity that is not further reduced by ceramide treatment.Coimmunoprecipitation studies reveal that the treatment with IGF-Iinduces the association of ERK with PKC- but not with PKC-.Ceramide treatment significantly inhibits the IGF-I-induced PKC-interaction with bioactive phosphorylated ERK. Ceramide also inhibitsIGF-I-induced PKC- association with Raf-1, an upstream kinase ofERK. Together, these studies demonstrate that ceramide exertsanti-mitogenic actions by limiting the ability of PKC- to form asignaling complex with Raf-1 and ERK.