The levels of messenger RNAs for the surfactant-associated proteins, SP-A, SP-B, and SP-C, have been examined in the developing rabbit lung in vivo. Northern blot analysis detected SP-C mRNA by day 22 of gestation (term 31 days) and SP-A mRNA and SP-B mRNA on day 26, while solution hybridization assays detected all three mRNAs on day 22 of gestation. Both techniques revealed that the mRNA levels increased rapidly during the last quarter of gestation. The mRNA levels determined by solution hybridization were highly correlated during development, with average molar ratios of 1.0:1.1:2.1 for SP-A, SP-B, and SP-C, respectively. We also examined the effect of accelerating fetal pulmonary maturation by maternal administration of either 17-beta-estradiol or betamethasone (9-alpha-fluoro-16-beta-methylprednisolone) on day 26 of gestation. These treatments increased SP-A mRNA levels 8- to 12-fold, resulting in levels 3- to 4-fold greater than in the adult. SP-B mRNA levels increased by approximately 2-fold to near adult levels, while SP-C mRNA was lowered somewhat by 17-beta-estradiol and significantly to less than half by betamethasone. No differences in the levels of surfactant apoprotein mRNAs or in choline incorporation into total or disaturated phosphatidylcholine were noted between male and female fetuses. These observations are consistent with the accepted view that the genes for the surfactant-associated proteins are independently regulated. However, the various factors affecting these mRNAs result in a coordination of mRNA levels during normal perinatal development.

The present communication documents attempts to produce the mature form of human surfactant-associated protein B (SP-B) by modification of the 5' and 3' regions of the cDNA and expression of the truncated cDNAs after insertion into the vector pKK223-3. The 5' end of a cDNA for human SP-B (1407 base pairs) was reconstructed through the ligation of synthetic oligonucleotides to an internal PstI site in the 5' region. This construction coded for the initiation of protein synthesis at a Met codon adjacent to a codon for the N-terminal Phe of the mature polypeptide. Variable amounts of the 3' end of the human SP-B cDNA were deleted with mung bean nuclease and exonuclease III. The resulting blunt-ended 3' fragments were then ligated to a synthetic oligonucleotide linker designed to create a stop codon. The modified 5' and 3' ends were ligated to a short PstI-BamHI fragment isolated from the SP-B cDNA and inserted into the expression vector pKK223-3. In vitro translation of sense mRNAs derived from the truncated SP-B cDNAs yielded oligopeptides of appropriate molecular weights, as indicated by urea - sodium dodecyl sulphate - polyacrylamide gel electrophoresis of either intact or immunoprecipitated reaction mixtures. Expression of SP-B in Escherichia coli was confirmed by Northern blot analysis for the mRNAs corresponding to the truncated cDNAs in appropriately transformed bacteria induced with the galactose analog isopropyl-beta-thiogalactoside. Western blot analysis using rabbit antisera prepared against bovine SP-B confirmed the presence of mature SP-B in lipid extracts of transformed E. coli, but the amounts were very small.(ABSTRACT TRUNCATED AT 250 WORDS)

The placenta from 30 women with diabetes mellitus were examined and weighed at delivery. Nineteen of these were from women with overt and eleven from women with gestational diabetes. Eleven placentae from normal pregnancies served as controls. There was no difference between the mean ± s.d. placental weight for the diabetic group and the control group (609 ± 148 versus 591 ± 93 g, NS). The mean placental weight ratios for the diabetic group and the control group were also similar (0.98 ± 0.23 versus 0.89 ± 0.15, NS). Moreover, there was no difference between the weights and weight ratios of placentae from women with overt (622 ± 173 g, 1.02 ± 0.27) and those with gestational diabetes (586 ± 90 g, versus 0.90 f 0.13). Placental weights correlated with birthweights (r = 0.70, P < 0.01) and with skinfold thickness measurements of the infants (r = 0.40, P < 0.05), but neither with gestational ages (r = 0.15, NS) nor with maternal glycosylated haemoglobin levels in the third trimester (r = 0.24, NS). Among the women with overt diabetes, placental weights were greater in those in White's class B and C than those in class D and R (689 ± 143 versus 530 ± 177 g; P < 0.05). In general, placentae from well controlled diabetic patients were not heavier than those from normal pregnant women, although there was an increase in placental weight in White's class B and C, as compared with those in class D and R.

The properties of natural bovine surfactant and its lipid extract have been examined with a pulsating bubble surfactometer which assesses the ability of surfactant lipids to adsorb to the air/liquid interface and reduce the surface tension to near 0 dynes/cm during dynamic compression. Studies conducted at 1 mg/ml phospholipid revealed that the surface activity (i.e., the ability to produce low surface tensions) of lipid extracts could be enhanced by incubating the sample at 37 degrees C for 120 min or by addition of CaCl2. In contrast, incubation at 37 degrees C only slightly improved the biophysical activity of natural surfactant and the addition of CaCl2 had a more modest effect than with lipid extracts. With 20 mM CaCl2, the surfactant activity of lipid extract surfactant was similar to that of natural surfactant. Incubation with EDTA reduced the biophysical activity of natural surfactant. Experiments in which increasing amounts of lipid extract were replaced by natural surfactant revealed that small amounts of natural surfactant enhanced the surfactant activity of lipid extract. The biophysical activity of lipid extract surfactant was also increased by the addition of soluble surfactant-associated protein-A (SP-A) (28-36 kDa) purified from natural bovine surfactant. These results indicate that SP-A (28-36 kDa) improves the surfactant activity of lipid extracts by enhancing the rate of adsorption and/or spreading of phospholipid at the air/liquid interface resulting in the formation of a stable lipid monolayer at lower bulk concentrations of either phospholipid or calcium.

ABSTRACT. Chloroform:methanol extracts of bovine pulmonary surfactant contain small hydrophobic proteins, designated surfactant-associated apoproteins 6,000 (SAP-6), but do not contain the major surfactant-associated 35,000-dalton glycoprotein, designated SAP-35. Examination of lipid extract surfactant on sodium dodecylsulfatepolyacrylamide gel electrophoresis revealed hydrophobic proteins with apparent molecular masses of 15,000, 7,000, and 3,500 daltons prior to reduction. After reduction, the 15,000-dalton species largely disappeared and was replaced by a 5,000-dalton species. In addition, the 7000-and 3500-dalton species exhibited a slightly enhanced mobility. Amino acid analysis demonstrated that SAP-6 possesses a more highly hydrophobic profile than SAP-35. Combining the protein-containing fractions from silicic acid chromatography of lipid extract with synthetic dipalmitoylphosphatidylcholine produced a reconstituted surfactant preparation which was just as active as lipid extract surfactant on a pulsating bubble surfactometer. The reconstituted surfactant contained SAP-6 but not SAP-35. Pressure-volume studies revealed that, at the optimal dose, reconstituted surfactant containing half the SAP-6 concentration of lipid extract exhibited similar effectiveness to lipid extract surfactant in promoting lung expansion with prematurely delivered rabbit fetuses of 27 days gestation. Reconstituted surfactant with an identical SAP-6 protein concentration as lipid extract possessed the same biological properties as the preparation with 1% SAP-6 protein. These studies support the view that an artificial surfactant composed of synthetic or semisynthetic lipids plus human SAP-6 produced via biotechnology could be useful for prevention and/or treatment of the neonatal respiratory distress syndrome.

To examine the role of parturition on lung maturation in sheep, we studied parameters of lung development in singleton fetuses treated with pulsatile adrenocorticotropic hormone or saline solution from day 127 or twin pregnancies in which one fetus only received pulsatile adrenocorticotropic hormone from day 127 until labor occurred. These parameters were compared with those of term fetuses (145 days). Pulsatile adrenocorticotropic hormone provoked labor in a mean (±SEM) of 102.6 ± 6.6 and 181.0 ± 18.0 hours in single and twin pregnancies, respectively. Adrenal/body weight ratios increased similarly in adrenocorticotropic hormone-treated single and twin fetuses at delivery, and basal cortisol levels were two- to threefold higher prepartum in adrenocorticotropic hormone-treated fetuses. Little change chaplasma cortisol levels occurred in singletons treated with saline solution or in twins not infused with adrenocorticotropic hormone. The lung weight/body weight was not altered in any group. Lung distensibility and stability were doubled to term values in fetuses treated with pulsatile adrenocorticotropic hormone compared with controls and untreated twins. Mean lavage phosphatidylcholine levels rose from 0.07 to 0.11 mg/gm in saline solution-treated or untreated fetuses to 0.20 to 0.23 mg/gm in pulsatile adrenocorticotropic hormone-treated singletons or twins, compared with 0.63 mg/gm at term. Phosphatidylcholine production increased from 0.51 dpm/gm/hr in saline solution-treated fetuses to 0.73 and 0.89 dpm/gm/hr in the single and twin pulsatile adrenocorticotropic hormone-infused fetuses, respectively; phosphatidylcholine production was 0.62 dpm/gm/hr in the non-infused twin. Lungs of twins treated with pulsatile adrenocorticotropic hormone were morphologically more mature than those of untreated twins. We conclude that fetal endocrine responses to exogenous adrenocorticotropic hormone, rather than the stimuli associated with labor per se, are responsible for lung maturation in the fetal sheep.

Lipid extracts of bovine pulmonary surfactant, which retain many of the biophysical characteristics of natural surfactant, contain approx. 98% lipid and 2% protein, as determined by amino acid analysis. Polyacry (amide/urea gel electrophoresis reveals that lipid extract surfactant contained a major apoprotein band with apparent Mr 3500 and minor apoprotein bands with apparent Mr 15000 and 7000. After reduction, the 15 kDa band disappears and is replaced by a prominent band with apparent Mr = 5000. Reduction also results in a relative diminution of the 7 kDa band and a relative increase in the intensity of the 3.5-kDa band. Edman degradation reveals two major peptide sequences which have been designated surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptide (N-terminal Leu) and a minor sequence designated surfactant-associated peptide (N-terminal Ile). The latter surfactant-associated peptide appears to be related to the N-terminal Leu peptide but lacks the terminal Leu. N-Terminal analysis by dansylation demonstrates that the 15 and 5 kDa (reduced) apoprotein species contain N-terminal Phe, Leu and Ile. The 3.5 and 7 kDa bands contain only N-terminal Leu and Ile. Chromatography of lipid extracts on silicic acid columns gives rise to fraction I, which contains protein and phosphatidylglycerol, and fraction II, which contains protein, phosphatidylglycerol and phosphatidylethanolamine. Fraction I was primarily composed of the 15-kDa apoproteins, while fraction II contained mainly the 3.5 and 7 kDa apoproteins. Both fractions exhibited biophysical activity after reconstitution with dipalmitoylphosphatidylcholine. These results indicate that lipid extracts contain an oligomer of 15 kDa containing surfactant-associated peptide (N-terminal Phe) and surfactant-associated peptides (N-terminal Leu or Ile) which interact through sulfhydryl and perhaps other bonds. Lipid extracts also contain 3.5 kDa monomers of surfactant-associated peptides with N-terminal Leu and N-terminal He which can dimerize through sulfhydryl and perhaps hydrophobic interactions.

Lipid extracts of bovine pulmonary surfactant, which exhibit biophysical and biological activity, contain two hydrophobic proteins which have been designated surfactant protein-B (SP-B) and SP-C. Amino terminal amino acid sequence analysis of whole lipid extracts and partially purified protein fractions gave rise to three sequences, two major and one minor. The first sequence, identified as a member of the SP-B family, extended for 60 amino acids beginning with an amino terminal phe. The second polypeptide, identified as a member of the SP-C family, sequenced for 35 amino acids and had a leu amino terminus. The third minor sequence corresponded to amino acids 2-9 of SP-C (N-leu) and was designated SP-C (N-ile). Sequence analysis of cyanogen bromide peptides derived from methyl isocyanate-blocked lipid extract material produced two peptides which extended the amino acid sequence of SP-B to residue 79, which appears to be a glycine.

A method for preparing a homogeneous population of undifferentiated cells from the fetal rabbit lung is described. This method utilizes enzymatic digestion, differential adhesion to remove fibroblasts and centrifugation on a discontinuous metrizamide gradient. Cells isolated by this procedure replicate in vitro in medium supplemented with carbon-stripped fetal bovine serum. Mitosis can also be stimulated by heat-inactivated medium conditioned by fetal lung fibroblasts. After confluence, exposure of these cells to 0.55 or 55 nM dexamethasone significantly increased the incorporation of [14C]choline into phosphatidylcholine. Lower concentrations of the drug also increased incorporation, but not significantly so. Addition of heat-inactivated fibroblast-conditioned medium produced a 25% increase in choline incorporation, but this was not significant. Furthermore, the presence of conditioned medium tended to reduced the response of the cells to dexamethasone. After confluence, lamellar inclusion bodies were present in more than 90% of those cells exposed to dexamethasone. These organelles were not observed in cell monolayers not exposed to the steroid. These cells did contain a few small electron-dense bodies. The latter may be immature multivesicular bodies.

The mural thickness of fetal stem arteries of 3rd order was assessed morphometrically in 50 placentae from each of the 'toxaemia', normal pregnancy and acute fetal distress groups. Several clinical maternal and fetal variables and the syncytial sprout proliferation of the placentae were correlated with the morphometric findings. The results show that: (1) there was a significant reduction in the ratio of lumen-to-whole-diameter of the fetal arteries in 'toxaemia' as compared with the two other groups; (2) the mean lumen-to-whole-diameter ratio also differed between regions of the placenta in all groups, the most marked reduction being in the parachorial region and the least prominent in the parabasal zone; (3) no significant differences in the mean diameter ratio were found among the three sub-groups of the toxaemic pregnancies, i.e., the preeclampsia, essential hypertension and renal disease group; and (4) there was an inverse relationship between the lumen-to-whole-diameter ratios and the syncytial sprout counts in the toxaemic group.

Natural surfactant possesses the ability to rapidly reduce the surface tension of a bubble pulsating at 20 cycles per min at 37°C to less than 30 dyn/cm at maximum radius and to less than 1 dyn/cm at minimum radius. The preparation of two artificial surfactant systems, containing either dipalmitoylphosphatidylcholine (DPPC) and phosphatidylethanolamine (PE) (5:5), or DPPC plus PE and phosphatidylglycerol (5:4:1 or 6:3:1), is described. Formation of artificial surfactants which mimic the essential properties of natural surfactant was correlated with the appearance of particles of aggregated lipids. The effects of lipid composition, calcium ion concentration, pH, temperature and mechanical agitation were determined. It is proposed that these artificial surfactant systems may produce a surface-active monolayer through the involvement of nonbilayer structures with properties similar to hexagonal HII phase.

Two fetuses of a patient affected with myotonic dystrophy were studied ultrasonically from 28 to 34 weeks' gestation. After a 1-hour observation period, an intravenous injection of 25 gm of 50% glucose solution was given to the mother. Fetal breathing movements were 0% during the control period and increased to only 10% at 90 minutes after the injection of glucose; the episode lasted approximately 30 minutes. The infants, who were delivered at 33 and 35 weeks, had generalized hypotonia, normal arterial cord blood gases, and died shortly after birth from pulmonary insufficiency, in spite of maximum ventilatory support. Postmortem pulmonary hypoplasia was confirmed by a lung weight/body weight ratio of less than 0.019. We postulate that fetal breathing activity and its response to the injection of glucose may be a potential clinical test by which normal fetuses can be differentiated from fetuses affected by neuromuscular disorders, including myotonic dystrophy.

The incorporation of radioactive glycerol into phosphatidylcholine by rat lung slices increased approximately 4-fold between 19 days gestation and the first day after birth. Increases were also observed with a number of other pulmonary lipids. The specific activities of a number of enzymes related to the biosynthesis of phosphatidylcholine and the acidic phospholipids, phosphatidylglycerol and phosphatidylinositol, were examined in whole homogenates and in the nuclear, mitochondrial, microsomal and cytosolic fractions. The specific activity profiles for a number of enzymatic markers for mitochondria and endoplasmic reticulum were also examined. The specific activity of phosphatidate cytidylyltransferase in the mitochondrial fraction was high during the perinatal period but fell in the adult. On the other hand, the specific activity of the microsomal fraction was low, compared to the mitochondrial fraction in early gestation, but increased near term and after birth, resulting in a specific activity in the adult which was greater than that observed with the mitochondria. Similar specific activity profiles were observed with the endoplasmic reticulum marker NADPH-cytochrome c reductase and with cholinephosphotransferase and inositol phosphatidyltransferase. Although the specific activity of glycerophosphate phosphatidyltransferase in the mitochondrial fraction also increased in late gestation and declined somewhat after birth, the specific activity of the microsomal fraction showed little alteration during development and remained low, compared to the mitochondria, even in the adult. Plots of total enzymatic activity per g lung were consistent with the view that the endoplasmic reticulum is the primary site for the biosynthesis of phosphatidylcholine, CDPdiacylglycerol and phosphatidylinositol, but the mitochondria possess the bulk of the enzymatic activity required for the formation of phosphatidylglycerophosphate and, therefore, phosphatidylglycerol. The basis for the unanticipated sedimentation of a considerable proportion of the activities of a number of those enzymes normally recognized as endoplasmic reticulum constituents with the mitochondria fraction during early development remains unresolved. It is clear that these changes in sedimentation properties could influence the results of experiments where a single subcellular fraction was examined.

The incorporation of radioactive choline into phosphatidylcholine and disaturated phosphatidylcholine in rat lung slices increased markedly before term and peaked after birth. The specific activity of cholinephosphate cytidylyltransferase in the microsomal fraction increased before birth but fell after delivery. The specific activity of this enzyme in the cytosol showed a marked increased at birth. The developmental profile for the total cytosolic activity per gram lung was similar to the pattern observed with choline incorporation. Although the specific activity of cholinephosphotransferase in the whole homogenate remained relatively constant throughout pulmonary maturation, there was a marked increase in the specific activity of this enzyme in the microsomal fraction at term. Similar findings were obtained with the microsomal marker NADPH-cytochrome c reductase. The basis of this disparity in specific activity profiles is being investigated further. The specific activity of lysophosphatidylcholine:lysophosphatidylcholine transacylase in rat lung homogenates increased during gestation but rose a further 10-fold between day 3 after birth and the adult. The specific activity of lysophosphatidylcholine:palmitoyl-CoA acyltransferase remained relatively constant throughout development. At term, the specific activity of the acylation enzyme was 10- to 15-fold greater than the specific activity of the transacylation enzyme. These observations are consistent with previous studies indicating that the accumulation of phosphatidylcholine and dipalmitoyl phosphatidylcholine during the perinatal period may be due to alterations in the activity of cholinephosphate cytidylyltransferase. Cholinephosphotransferase could also play a regulatory role. The formation of dipalmitoyl phosphatidylcholine appears to occur via the acylation, rather than the transacylation pathway.

1.1. Administration of estradiol-17β to pregnant rabbits at 25 days gestation (term, 31 days) resulted in a significant increase in the incorporation of [14C]-choline, but not [14C]ethanolamine, into the lipids of fetal lung slices. The incorporation of [35S]methionine was not affected.2.2. Enzymatic assays conducted in vitro revealed no significant effect on either the activities of several enzyme markers for subcellular organelles, the activities of the enzymes responsible for the production of phosphatidylglycerol and phosphatidylinositol, membrane-bound or aqueously dispersed phosphatidate-dependent phosphatidic acid phosphohydrolase activities or the activities of the auxiliary enzymes responsible for the synthesis of dipalmitoylphosphatidylcholine.3.3. The activity of the enzymes involved in the choline pathway for the de novo biosynthesis of phosphatidylcholine were not significantly altered except for a 66% increase in the CTP:cholinephosphate cytidylyltransferase activity assayed in the cytosol. The addition of phosphatidylglycerol stimulated cholinephosphate cytidylyltransferase activity approx. 3-fold. However, in the presence of this lipid, the activities in cytosol from control and treated fetuses were similar, indicating that the increased activity noted in the absence of phosphatidylglycerol was due to an activation of existing cytidylyltransferase activity rather than an increase in total enzyme units.4.4. Estrogen treatment of the does was also associated with a marked decrease in the levels of cholinephosphate in fetal lung and significant increases in the levels of CDPcholine and phosphatidylcholine. These alterations in pool size are consistent with an increase in the activity of cholinephosphate cytidylyltransferase in vivo. The results suggest that cholinephosphate cytidylyltransferase may catalyse an important rate-determining reaction in the synthesis of phosphatidylcholine in fetal lung. The data also support the view that the reaction catalysed by CDPcholine: diacylglycerol cholinephosphotransferase also has a regulatory role during development.

Two cases are presented in which the effects of blunt trauma to a pregnant woman's abdomen were apparently minor but resulted in fetal death. Blunt trauma may result in serious injury to the fetus or the placenta. Three-point restraint systems should be worn by pregnant women travelling in automobiles to minimize the risks to mother and fetus. Awareness of the potential for injury in these circumstances is essential to reduce the risks to the fetus.

The incorporation of intraperitoneally injected 3H-glycerol and 14C-palmitate into the lipids of lung, liver and brain was examined in premature rabbits (27 days gestation, term 31) both in utero and in control or surfactant-treated neonates. With the lung, there was an increased incorporation after delivery, especially with the surfactant-treated pups. The incorporation of 3H-glycerol into liver lipids was markedly reduced after birth, particularly with the nontreated controls. The liver:lung ratios of the incorporations were decreased after delivery. In all three tissues, the relative incorporations into the individual lipids were not affected to any great extent by birth or surfactnat treatment. In lung, the disaturated and monoenoic species, respectively, of phosphatidylcholine accounted for the major proportion of the radioactivity. After birth there was a small increase in the labelling of the monoenoic species at the expense of the disaturated species with both control and surfactant-treated neonates. These observations support the proposal that surfactant treatment of the prematurely delivered neonate could be used prophylactically for the prevention of the respiratory distress syndrome.