Studies on lung surfactant replacement in respiratory distress syndrome. Rapid film formation from binary mixed liposomes

Binary mixed liposomes were prepared from dipalmitoylphosphatidylcholine (DPPC) and a minor compound, e.g., egg phosphatidylglycerol (PG) at a ratio of 9:1. Using different preparative techniques, large unilamellar vesicles (LUV), small unilamellar vesicles (SUV) or multilamellar vesicles (MLV) were obtained and were studied with an electron microscope for morphology, with a Wilhelmy balance for spreading and surface tension lowering potential, and in the surfactant-depleted isolated rat lung for their ability to restore expiratory lung capacity. Only the simultaneous investigation of phospholipids by negative staining and thin sectioning allows unequivocal classification of liposomes. The surface-active structures prepared with the technique of Bangham et al. (Bangham, A.D., Hill, M.W. and Miller, N.G.A. (1974) in Methods in Membrane Biology (Korn, E., ed.), Vol. 1, pp. 1-68, Plenum Press, New York) at room temperature are LUV. LUV containing DPPC:PG at a ratio of 9:1 rapidly spread to a film with high surface tension lowering potential. Within 5 min after injection into the subphase they rise to the surface and form a film at the air/liquid interface able to lower the surface tension to less than 1 mN/m at compression. SUV of the same chemical composition, however, are immediately surface-active only when spread directly onto the surface. MLV exhibit poor surface activity. LUV or pure DPPC, applied onto the surface, are weakly surface active within 5 min. DPPC vesicles injected into the subphase at 37 degrees C do not adsorb to any film with surface tension lowering potential in this time. The minor compounds PE, PI, PS, PA, lysoPC enable DPPC to form surface-active films after application on saline at 37 degrees C. Removal of surfactant decreases the expiratory lung capacity of the isolated rat lung from 49.7 to 12.4% at 4 cmH2O. After substitution with natural surfactant, the expiratory lung capacity is twice that of the washed lung (25.9%), but the original distensibility of the native lung is not restituted. The effect of LUV containing DPPC:PG at a ratio of 9:1 is also remarkable (21.2%).     

Inhibition of beta-1,4-Glucan Biosynthesis by Deoxyglucose : The Effect on the Glucosylation of Lipid Intermediates

GDP- and UDP-deoxyglucose inhibit the incorporation of glucose from UDP-glucose into dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides. GDP-deoxyglucose inhibits by competing with the physiological nucleotide sugars for dolichyl phosphate, and dolichyl phosphate deoxyglucose is formed. This inhibition is reversed by excess of dolichyl phosphate. UDP-deoxyglucose does not give rise to a lipid-linked derivative, and inhibition by this analog is not reversed by dolichyl phosphate. The UDP- and GDP-derivatives of deoxyglucose inhibit the incorporation of glucose into glucose-containing glycoproteins. This effect seems to be the result of the inhibition of lipid intermediates glucosylation and is comparable to the effect produced by coumarin. Cellulose synthetase activity is not affected by UDP- or GDP-deoxyglucose. On the other hand, deoxyglucose inhibits the formation of β-1,4-glucans in vivo.     

Disappearance and reformation of the nuclear lamina structure during specific stages of meiosis in oocytes

The nuclear lamina is a rigid, proteinaceous layer underlying the inner nuclear membrane of eucaryotic cells. It is present in somatic cell nuclei, disappears during mitosis, and is absent from male meiotic cells. We have investigated the disappearance and reformation of the nuclear lamina during meiosis in oocytes, using immunofluorescence and electron microscopy. We find that the status of the nuclear lamina during meiosis of oocytes differs from the reversible depolymerization seen in mitosis in two respects. First, the lamina disappears during meiotic prophase without affecting the structure of the nuclear membranes or the nuclear pores. Second, the proteins of the dissociated lamina are undetectable by immunological methods in pachytene oocytes, whereas they persist in the cytoplasm during mitosis.     

Oriented axon outgrowth from avian embryonic retinae in culture

The neural retina of avian embryos was spread on a membrane filter and cut in any desired orientation. Strips cut across the retina of 4- to 7-day chick or 3- to 6-day quail embryos were explanted onto collagen gels. Vigorous neurite outgrowth was seen for about 3 days, by which time many neurites were 3 mm long. Horseradish peroxidase (HRP) labeling showed that the cells producing the neurites were large and formed a layer near the inner limiting membrane, indicating that the neurites in vitro were axons of retinal ganglion cells. The size of the neurite population and the regions from which neurites emerged vaired with the donor age, while most neurites sprouted from the side of the explant formerly closest to the optic fissure. This pattern closely resembled that of axon growth in the normal retina, as revealed by SEM, silver staining, and HRP labeling. Mitotic inhibitors (Ara-C and FUdR) did not alter the neurite outgrowth. Pretreatment of retinae with trypsin or collagenase did not disorganize axons at the time of explantation, but tended to equalize neurite emergence on each side of the retinal strips. We suggest that microenvironmental factors, especially the enzyme-labile inner limiting membrane, are important for axon guidance in the retina.     

Steroid derivatives

Microbial transformation of some steroid 19-hydroxy compounds and 1(10),5-dienes, substituted by a hydroxyl group in position 3, by the action ofProactinomyces globerulus, yielded substances with aromatic A ring. Substrates with 17-keto group underwent simultaneously a partial reduction resulting in the formation of 17β-hydroxyl.     

Limestone gravel as growth medium in hydroponics

Summary Plants were grown in gravel beds of basalt or limestone. With the standard nutrient solution yields for the plants grown in the limestone gravel were very much reduced and the plants were chlorotic. This chlorosis and reduced yields remained even if the gravel was acid or water washed beforehand, or if it was pretreated in high phosphate concentrations.High yields, comparable to those obtained in basalt beds were obtained if phosphate and iron were added in small amounts (0.1mM P, 1 mg/1 Fe) at each irrigation. This technique to avoid lime induced chlorosis can be easily and economically accomplished in hydroponics. It proved successful here with lettuce, cucumber, tomato and eggplants.     

Recovery of Adrenocortical Function during Long-term Treatment with Corticosteroids

The recovery of adrenocortical function during very slow withdrawal of corticosteroids was studied in a homogeneous group of patients suffering from sarcoidosis. All patients had been treated with gradually decreasing doses of prednisone for at least two years. The initial dose had been 40 mg. daily in all cases. Determination of the cortisol production rate and of plasma fluorogenic corticosteroids was done under basal conditions and after tetracosactrin stimulation. There was good correlation between cortisol production rate and plasma fluorogenic corticosteroids throughout all the tests. Cortisol production rate and plasma fluorogenic corticosteroids started to rise when the dosage of prednisone was lowered to 7·5 mg. daily and reached normal values when the dosage was reduced to 2·5 mg. The response to tetracosactrin began to increase at the same dosage level, but was not normal at 2·5 mg., or when prednisone treatment was stopped. At a dosage level of 7·5 mg. of prednisone plasma fluorogenic corticosteroids already showed a nyctohemeral rhythm.It may be calculated that even very low dosages of prednisone given during the last stage of a treatment schedule enhance total corticosteroid activity beyond the normal level, which would account for their therapeutic value.     

Über die Feinstruktur der menschlichen Cornea,mit besonderer Berücksichtigung des Problems der Transparenz

Zusammenfassung 3 normale, gesunde, menschliche Corneae wurden unmittelbar nach der Augenenukleation entnommen und elektronenmikroskopisch untersucht. Die Kollagenfibrillen wurden auf eine symmetrische Anordnung in Stromaquerschnitten untersucht. Die Lage der Fibrillen in 100 geordneten Regionen wurde gemessen und in ein Diagramm eingetragen. Eine Symmetrie, die eine Gittertheorie der Durchsichtigkeit stützen könnte, wurde nicht gefunden. Der Durchmesser der Fibrillen beträgt 23–27 m. Der Abstand der Fibrillen voneinander liegt zwischen 10 und 40 m. Damit beträgt der Volumenanteil der kollagenen Fibrillen 20%, an manchen Stellen weniger. So ist der tatsächliche Volumenanteil der Kollagenfibrillen nur 7% des Stroma.

---

Summary 3 normal human corneaes were excised immediately after enucleation of the eyes and observed in the electron microscope. The collagen fibrils have been examinted for symmetrical arrangement in transverse sections of the stroma. The position of the fibrils in 100 undisturbed regions was measured and registered in a diagram. No symmetry was found to support the lattice theories of transparency. The diameter of the fibrils is 23–27 m. The distance between the fibrils is 10–40 m. That means 20% volume collagen in the stroma, in some areas less. So the collagen fibrils occupy only 7% in the entire stroma.

---

---

Herrn Prof. Dr. Ing., Dr. med. h. c., Dr. phys. h. c. E. Ruska zum 60. Geburtstag gewidmet.     

Elektronenmikroskopische Untersuchungen an Lysosomen im Blastem der Extremitätenknospe der Ratte

Zusammenfassung Die Extremitätenknospen und die Schwanzknospe von Rattenembryonen enthalten ein mesenchymales Zellretikulum, in dessen Achse sich die Zellen polygonal abrunden und zu einem Blastem zusammenlegen. In den umgebenden mesenchymalen Zellen kommen keine Lysosomen vor. Dagegen enthalten die Zellen des zentralen Blastems Lysosomen. Die primären Lysosomen sind cytoplasmatische Granula mit einem Durchmesser von 0,1–0,2 und einem elektronendichten granulären Inhalt. Auffällig ist das Vorkommen von multivesikulären Einschlußkörpern in der Nähe der Golgizone. Wegen ihrer Funktion können sie auch zu den primären Lysosomen gerechnet werden. Die häufigsten Lysosomen sind runde Cytolysosomen (Durchmesser von 0,5–2 ), die von einer Membran umgeben sind und polymorphen Inhalt besitzen. Dicht gepackte Granula, die wie zusammengeflossene Ribosomen aussehen, kommen vor. Außerdem liegen andere Zellorganellen (Mitochondrien, Membranstrukturen) in den sekundären Lysosomen. Schließlich kommen auch Myelinfiguren, allein oder mit anderen Strukturen kombiniert, in ihnen vor. Die verschiedenen Stadien dieser Lysosomen sind der Ausdruck für eine partielle Autodigestion von Zellorganellen im Blastem. Eine Autodigestion von Ribosomen könnte eine Bedeutung haben. Nach dieser Autodigestion der alten Information und der Ribosomen beginnt nämlich eine Zelldifferenzierung zu Vorknorpel mit neuer Information und neuen Ribosomen für die jetzt neu auftretende Zwischensubstanz. Die beschriebenen Lysosomen zeigen eine positive elektronenmikroskopisch nachweisbare Reaktion auf saure Phosphatase.

---

Summary The limb buds and the tail bud of rat embryos contain a mesenchymal cellular reticulum. In its axis the cells become polygonal and group to a blastema. There are no lysosomes in the cells of the surrounding mesenchyma. The cells of the central blastema, however, possess lysosomes. The primary lysosomes are cytoplasmic granules with a diameter of 0,1–0,2 . Their granular contents are electron dense. There is a striking occurrence of multivesiculated bodies near the Golgi zone. For their function they may be counted to the primary lysosomes. The most frequent lysosomes are the cytolysosomes. These are of circular shape with a diameter of 0,5–2 and are enclosed by a membrane, their contents are polymorphic. Tightly stacked granules looking like conglomerated ribosomes can be found. Apart from these other cell organelles like mitochondria and membrane structures lie in the secondary lysosomes. Finally, there are also myelin figures alone or combined with other structures in the lysosomes. The different stages of these lysosomes are the result of a partial autodigestion of cell organelles within the blastema. The autodigestion of ribosomes may be important. After this autodigestion of the old template and ribosomes a new phase of cyto-differentiation to precartilage with new template and ribosomes begins. The lysosomes show a positive reaction for acid phosphatase.