Lung surfactant synthesis: a Ca++-dependent microsomal phospholipase A2 in the lung.

We present the first direct evidence for a highly active, Ca⁺⁺-dependent phospholipase A₂ in the microsomal fraction of rat lung homogenate. Several previously reported studies from other laboratories strongly implicate this enzyme as a key metabolic step in the biosynthesis of dipalmitoyl lecithin, the primary component of pulmonary surfactant. In the present study, stoichiometric amounts of [³H]lysophosphatidylethanolamine and [¹⁴C]fatty acid were released during incubation of 1-[9, 10-³H]palmitoyl-2-sn-[1′-¹⁴C]linoleoyl phosphatidylethanolamine with the lung microsomal fraction. Marker enzyme measurements showed that the microsomal activity cannot be due to contamination with mitochondria, which also show phospholipase A₂ in both lung and liver. In contrast, liver microsomes show predominantly a phospholipase A₁ activity.     

Potential and realised fecundity in the bush fly, Musca vetustissima under favourable and unfavourable protein-feeding regimes

The number of ovarioles in female Musca vetustissima (potential fecundity/ovarian cycle) is positively correlated with fly size (headwidth) and potential fecundity does not vary between successive ovarian cycles. Field females of average headwidth (2 mm) have a mean potential fecundity of 25 eggs/cycle. Females are anautogenous, needing to ingest protein-rich material in order to mature their oocytes. Females that obtain insufficient protein material for maturation of full egg complements may cease ovarian development before or during early vitellogenesis, or resorb some of their oocytes and mature the remainder. Field females consistently matured in excess of 85% of their egg complements, indicating that female M. vetustissima have ready access to protein-rich material in the field.

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Résumé Le nombre d'ovarioles des Musca vetustissima femelles (fécondité potentielle/cycle ovarien) est lié directement à la taille de la mouche (largeur de la tête); la fécondité potentielle ne varie pas entre les cycles ovariens successifs. Les femelles de la nature, d'une largeur de tête moyenne (2 mm) ont une fécondité potentielle moyenne de 25 oeufs par cycle. Les femelles anautogènes, ont besoin d'un repas riche en protéines pour produire des oeufs mûrs. Les femelles qui n'obtiennent pas la quantité suffisante de protéines pour permettre la maturation d'un lot complet d'oeufs peuvent interrompre leur développement avant ou pendant le début de la vitellogenèse, ou résorber certains ovocytes tout en conduisant à maturité le reste. Les femelles de la nature conduisent a maturité 85% de leurs lots d'oeufs, montrant ainsi que les femelles de M. vetustissima y ont un accès facile à des aliments riches en protéines.

     

Potential dependence of the “electrically silent” anion exchange across the plasma membrane ofXenopus oocytes mediated by the band-3 protein of mouse red blood cells

Summary Mouse erythroid band-3 protein was incorporated into the plasma membrane ofXenopus oocytes by microinjection of poly(A)⁺-mRNA from spleens of anemic mice. Subsequently, the efflux of microinjected³⁶Cl was continuously followed in single oocytes in a perfusion chamber the bottom of which was formed by the window of a Geiger-Müller tube. During the flux measurements, the membrane potential was clamped to different holding potentials. The efflux increased over the voltage range of –10 to –100 mV by a factor of about 1.5. Since the membrane potential cannot act as a driving force of anion exchange, it is suggested that the observed slight potential dependence is related to a recruitment of the anion-loaded transport protein by the electrical field, thereby changing the steady-state distribution between inwardly and outwardly facing anion binding sites of the transport molecules.The experimental data are discussed in terms of ping-pong kinetics, assuming that the potential dependence is primarily due to an effect of the electrical field in the membrane on the ratelimiting interconversion of inwardly and outwardly oriented anion binding sites. The results are compatible with the assumption that in the oocyte membrane the substrate-loaded band-3 molecules are preferentially inwardly oriented, and that the transition from the inwardly to the outwardly oriented conformation is associated with a reorientation of an effective charge of 0.1 elementary charge.During progesterone-induced maturation of the oocytes, several endogenous transport systems change their activity drastically. The mouse band-3 protein in the oocyte membrane also undergoes activity changes; however, these changes do not seem to involve direct regulation by specific metabolic processes. They can be explained as a consequence of the depolarization of the membrane potential associated with the maturation process.     

Regulation of processing of a plant glycoprotein in the Golgi complex: A comparative study usingXenopus oocytes

The synthesis of phytohemagglutinin (PHA), the major seed lectin ofPhaseolus vulgaris, was investigated inXenopus oocytes injected with RNA isolated from developing bean cotyledons. As is the case for normal PHA, oocyte-synthesized PHA polypeptides were found to contain two asparagine-linked oligosaccharide chains, one of which was of the high-mannose type and the other one of the Golgi-modified type, being largely resistant to endo--N-acetylglucosaminidase H digestion and containing fucose. The modified oligosaccharide chain of oocyte-synthesized PHA appeared to be much larger and more heterogeneous with respect to the modified chain normally present on PHA. When the oocytes were injected with purified mRNA for PHA, isolated by hybrid-selection using a PHA complementary-DNA clone, the results were the same as those obtained by injecting total cotyledonary RNA. On the whole, these results indicate that plant glycoproteins are directed to the Golgi complex even when synthesized in an animal cell, and that correct sorting of the oligosaccharide chains to be processed is independent of the cell-type in which protein synthesis occurs. The form of processing is however cell-type specific.Abbreviations endo H endo H--N-acetylglucosaminidase H - ER endoplasmic reticulum - PHA phytohemagglutinin - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the adenohypophysis in the monkeys Macaca irus and Cercopithecus aethiops

Summary With the use of an antibody against bovine S-100 protein, it was possible to reveal a characteristic cell type in the pars distalis and the pars tuberalis of the monkey Macaca irus. In the adenohypophysis of Cercopithecus aethiops, labeled cells were present in the pars distalis, pars tuberalis, and pars intermedia. These cells, so-called folliculo-stellate cells, were found in all pituitaries studied. Surprisingly, an antibody against human S-100 protein did not label the stellate cells of the adenohypophysis. However, in Macaca irus, this antibody gave a strong positive reaction with various other cell types (interstitial cells of the pineal gland, Müller cells of the retina, autonomic ganglionic cells, glial cells of the central nervous system, Schwann cells, Bergmann glia of the cerebellum, fat cells, reticular cells of lymphoid organs). By use of double immunoenzymatic labeling, it was evident that stellate cells are spatially related either to somatotropes, prolactin cells, corticotropes, or to glycoprotein-containing cells. Thus, a specific relationship to a particular endocrine-cell type could not be observed.Dr. M.P. Dubois died in Tours (France) on March 30, 1986, aged 65     

Freeze-fracture observations of unfertilized and fertilized hamster oocytes with special reference to the use of lipid probes

Membrane and cytoplasmic changes were observed after in vitro fertilization of hamster oocytes by examining freeze-fracture replicas. The density of intramembranous particles on areas of membrane between microvilli increased following fertilization. Although the intramembranous particle density of microvilli is higher than that on the intermicrovillar membrane of unfertilized eggs, it did not change significantly after fertilization. Cytoplasmic changes in the Golgi complex and mitochondria upon fertilization indicate a change in cellular activity. Lipid binding probes were applied to the oocyte membranes in order to study the distribution of specific lipids before and after fertilization. Probes included the B-hydroxy-steroid complexing molecules, filipin and tomatin, and an anionic lipid binding antibiotic, polymyxin B. Both tomatin and filipin complex with steroids in the P and E faces of the plasma membrane (including the polar bodies), cortical granules and vesicles deeper in the cytoplasm, and the Golgi complex, leaving mitochondria, pronuclei, endoplasmic reticulum, and the majority of vesicles unlabeled. Polymyxin B binding is dependent on its application before or after fixation or in association with EGTA. With its application we detected both minor membrane perturbations of wrinkles and particle redistributions and major perturbations of vesicle fusions, the formation of blebs, and the loss of membrane morphology. Neither the distribution nor apparent quantity of these probes changed overall following fertilization, but this impression does not include specific sites of sperm-egg fusion.     

Mouse oocyte maturation modulated by a granulosa cell factor and by heparin and heparan sulfate

Oocyte maturation-preventing factor (OMPF) was extracted from bovine granulosa cells with a buffer containing 1 M urea and 5 mM EDTA. OMPF was partially purified by gel filtration on Sephadex G25 and by reversed-phase high performance liquid chromatography. The maturation-preventing activity of purified fractions was determined by measuring their capacity to block the spontaneous dissolution of the germinal vesicle (GVBD) of isolated cumulus-enclosed mouse oocytes. Hyaluronic acid, chondroitin sulfate, heparin, heparan sulfate, keratan sulfate, and dextran sulfate at concentrations of 500 μg/ml did not affect the frequency of GVBD of isolated mouse oocytes. Heparin and heparan sulfate, however, blocked the inhibitory effect of OMPF, whereas the inhibition of GVBD induced with dibutyryl cAMP, forskolin, W7 (calmodulin antagonist), and 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) was not blocked. OMPF was eluted in the adsorbed fraction when chromatographed on heparin-Agarose, showing interaction of OMPF with heparin. The present results suggest that the glycosaminoglycan matrix may influence OMPF action on oocytes.     

Giant eosinophil colonies from cultures of bone marrow cells

Summary To date, the small size and slow growth of eosinophil colonies in vitro has hampered study of cloned eosinophils. We found enhanced eosinophil colony size and numbers in methylcellulose cultures of bone marrow cells utilizing defined supplemented bovine calf serum (DSBCS) in combination with EL4 conditioned medium (EL4-CM). At days 9, 16 and 23 significantly more eosinophil colonies and more cells/colony were present in cultures incubated with DSBCS/EL4-CM than in cultures incubated with fetal calf serum/EL4-CM. The ability to generate large numbers of eosinophils in vitro should facilitate study of cloned eosinophils. Supported in part by a grant from the National Institutes of Health, AI 20416, and by the Mayo Foundation. Editor's statement Previous approaches to in vitro culture of eosinophils generally have achieved, at best, mixed cultures of colonies of these cells and granulocyte-macrophage colonies. The improved culture methods described in this report produce more homogeneous eosinophil cultures and larger colonies of these cells. The procedure employs EL4 murine thymoma-conditioned medium, which apparently contains eosinophil colony-stimulating activity in the absence of granulocyte-macrophage colony-stimulating activity. David W. Barnes     

Phototoxicity of phenothiazine derivatives. I. Inactivating and mutagenic effects on bacteriophage φX174

Inactivation of φX174 bacteriophages as a function of the irradiation time in the near-UV and in the presence of triflupromazine (TFPZ), promazine (PZ), chlorpromazine (CPZ) or methoxypromazine (MTPZ) proceeds according to single hit kinetics. Acepromazine (ACPZ) has no significant activity. At low concentrations (0.1 mM) TFPZ and PZ are the most active compounds. Higher concentrations (up to 5 mM) result in a protective effect by these two compounds but cause increased inactivation rates in the case of MTPZ or CPZ. Photoinactivation mediated by TFPZ or CPZ increases the reversion frequency of a φXamber mutant. Neither MTPZ nor PZ sensitization induces mutagenesis. The effect of NaN₃ on the phage inactivation rate varies depending upon both the sensitizer and the concentration of the quencher. Phage inactivation in an N₂ atmosphere is measurable only in the presence of high concentrations of CPZ and MTPZ. The drugs do not show any selectivity for calf thymus DNA or bovine serum albumin, at least as measured by dialysis equilibrium experiments.