Jeanne M. Snyder

A COMPARISON OF HUMAN AMNION TISSUE AND AMNION CELLS IN PRIMARY CULTURE BY MORPHOLOGICAL AND BIOCHEMICAL CRITERIA

SummaryProstaglandins, which are believed to mediate the initiation or maintenance of human labor or both, are synthesized from arachidonic acid. We have shown previously that the arachidonic acid content of two glycerophospholipids (diacyl phosphatidylethanolamine and phosphatidylinositol) in amnion tissue obtained during early labor is decreased compared with that in amnion tissue obtained before labor commenced. We and others have demonstrated that amnion tissue synthesizes prostaglandin E2 from exogenous and endogenous arachidonic acid. Thus, the amnion may be a source of prostaglandins involved in the initiation of parturition. We have investigated whether amnion cells in monolayer culture could be utilized as an in vitro model system for the study of glycerophospholipid and arachidonic acid metabolism in amnion. Several morphological biochemical, and enzymatic characteristics of amnion cells in culture were compared with those of amnion tissue. Morphologically, the amnion cells in culture and amnion tissue are similar. The lipid composition and arachidonic acid content of lipid fractions of amnion cells in culture and of amnion tissue also are similar. The specific activities of phospholipases A2 and C, the enzymes that initiate and thus probably regulate the release of arachidonic acid from phosphatidylethanolamine and phosphatidylinositol, are lower in amnion cells in culture than in amnion tissue obtained from term placentas. The specific activities of diacylglycerol lipase, monoacylglycerol lipase, and diacylglycerol kinase, enzymes that catalyze the release of arachidonic acid from diacylglycerol produced by the action of phospholipase C on phosphatidylinositol, are similar in amnion cells in culture and in amnion tissue. Therefore, we conclude that, based on morphology, lipid composition and enzymatic activities amnion cells in primary culture seem to be an appropriate in vitro model system for the investigation of the regulation of arachidonic acid metabolism in amnion.

Surfactant protein A (SP-A): the alveolus and beyond

Surfactant protein A (SP‐A) is the major protein component of pulmonary surfactant, a material secreted by the alveolar type II cell that reduces surface tension at the alveolar air‐liquid interface. The function of SP‐A in the alveolus is to facilitate the surface tension‐lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on surfactant function. It has also been shown that SP‐A modulates host response to microbes and particulates at the level of the alveolus. More recently, several investigators have reported that pulmonary surfactant phospholipids and SP‐A are present in nonalveolar pulmonary sites as well as in other organs of the body. We describe the structure and possible functions of alveolar SP‐A as well as the sites of extra‐alveolar SP‐A expression and the possible functions of SP‐A in these sites.—Khubchandani, K. R., Snyder, J. M. Surfactant protein A (SP‐A): the alveolus and beyond. FASEB J. 15, 59–69 (2001)

Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding

Dehydroepiandrosterone (DHEA) treatment is effective in the prevention of various genetic and induced disorders of mice and rats. In studies designed to define some of the basic mechanisms that underline the beneficial chemopreventive effects exerted by the action of this steroid, we found that the liver undergoes profound changes that result in: (i) hepatomegaly; (ii) color change from pink to mahogany; (iii) proliferation of peroxisomes; (iv) increased cross-sectional area and volume density of peroxisomes; (v) increased or decreased number of mitochondria per cell; (vi) decreased mitochondrial cross-sectional area; (vii) marked induction of the peroxisomal bifunctional protein enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase; (viii) increased activities of enoyl-CoA hydratase and other peroxisomal enzymes assayed in this study, viz. catalase, carnitine acetyl-CoA transferase, carnitine octanoyl-CoA transferase, and urate oxidase; and (ix) increased activity of mitochondrial carnitine palmitoyl-CoA transferase. In addition, feeding DHEA to mice resulted in increased plasma cholesterol levels in two strains of mice evaluated in this study, and either slightly decreased or markedly increased plasma triglyceride levels, depending on the strain. Whether liver peroxisome proliferation, induced by DHEA feeding to mice and rats, plays a role in the chemopreventive effects elicited by this steroid remains to be established.

Maintenance and characterization of human myometrial smooth muscle cells in monolayer culture

SummaryHuman myometrial cells were dispersed from uterine tissue by limited enzymatic digestion of myometrium that was obtained at the time of hysterectomy. The dispersed myometrial cells that are obtained in this manner can be maintained in monolayer culture in the presence of medium that contains fetal bovine serum. In primary culture, as well as after passage, the characteristics of these cells are morphologically and biochemically similar to those of smooth muscle cells and myometrial tissue.

Transient surfactant protein B deficiency in a term infant with severe respiratory failure

A 38-day-old male infant with persistent pulmonary hypertension and respiratory failure since birth was found to have a complete absence of surfactant protein B (SP-B) along with an aberrant form of SP-C in his tracheal aspirate fluid, findings consistent with the diagnosis of hereditary SP-B deficiency. Surprisingly, SP-B and SP-B messenger ribonucleic acid were present in lung biopsy tissue. However, DNA sequence analysis demonstrated a point mutation in exon 5 of one of the SP-B gene alleles. The infants mother was found to be a carrier of this mutation. The infants other SP-B allele did not differ from the published DNA sequence for the SP-B gene. We conclude that this patient had a transient deficiency of SP-B, in contrast to that of previously described infants with irreversible respiratory failure caused by hereditary SP-B deficiency. We recommend that infants with suspected SP-B deficiency have serial analysis of tracheal fluid samples for both SP-B and SP-C before lung biopsy, along with genetic analysis for the known SP-B mutations. We speculate that the new mutation found in one of this patients SP-B genes was in part responsible for the transient deficiency of SP-B.

The concentration of the 35-kDa surfactant apoprotein in amniotic fluid from normal and diabetic pregnancies

ABSTRACT: A specific, enzyme-linked immunoabsorbent assay was used to determine the concentration of the 35,000 mol wt surfactant apoprotein (SP-A) in samples of amniotic fluid obtained from nondiabetic (n = 358) and diabetic (n = 29) women. The enzyme-linked immunoabsorbent assay was performed with rabbit antibodies directed against SP-A present in lavage fluid from a patient with alveolar proteinosis. Amniotic fluid SP-A concentrations increased as a function of gestational age, from <3 μg/ml at 30–31 wk to 24 μg/ml at 40–41 wk, and were positively correlated with the lecithin to sphingomyelin ratio (p < 0.01). SP-A concentrations also increased as a function of gestational age in shake test positive samples (p < 0.05), but were unchanged in shake test-negative samples. There was no difference in the surfactant apoprotein concentration of male compared with female fetuses at any gestational age. In amniotic fluid obtained from 20 diabetic women, SP-A levels were significantly less than in nondiabetic pregnancies that were matched for gestational age and sex of the fetus (p < 0.05). The SP-A concentrations in amniotic fluids obtained from nine women who were diabetic and hypertensive and from 10 hypertensive women were not different from matched controls. The relationships described above were valid whether the SP-A concentration was expressed per mg protein or per ml amniotic fluid. These data are suggestive that the concentration of amniotic fluid SP-A is decreased in diabetic pregnancies.

Alveolarization in Retinoic Acid Receptor-β–Deficient Mice

Retinoids bind to nuclear receptors [retinoic acid receptors (RARs) and retinoid X receptors]. RARβ, one of three isoforms of RARs (α, β, and γ), is expressed in the fetal and adult lung. We hypothesized that RARβ plays a role in alveolarization. Using morphometric analysis, we determined that there was a significant increase in the volume density of airspace in the alveolar region of the lung at 28, 42, and 56 d postnatal age in RARβ null mice when compared with wild-type controls. The mean cord length of the respiratory airspaces was increased in RARβ null animals at 42 d postnatal age. Respiratory gas-exchange surface area per unit lung volume was significantly decreased in RARβ null animals at 28, 42, and 56 d postnatal age. In addition, alveolar ducts tended to comprise a greater proportion of the lung airspaces in the RARβ null mice. The RARβ null mice also had impaired respiratory function when compared with wild-type control mice. There was no effect of RARβ gene deletion on lung platelet-derived growth factor (PDGF) receptor α mRNA levels in postnatal lung tissue at several postnatal ages. However PDGF-A protein levels were significantly lower in the RARβ null mice than in wild-type controls. Thus, deletion of the RARβ gene impairs the formation of the distal airspaces during the postnatal phase of lung maturation in mice via a pathway that may involve PDGF-A.

Glucocorticoid Metabolism in the Human Fetal Lung: Implications for Lung Development and the Pulmonary Surfactant System

It has been nearly 35 years since Liggins and Howie first reported the benefits of antenatal glucocorticoid (GC) treatment to promote the maturation of the human fetal lung, and nearly that long since Pasqualini and colleagues demonstrated that the human fetal lung actively metabolizes GCs. Since that time, our understanding of the effects of GCs on fetal lung maturation and pulmonary surfactant production has increased dramatically. Similarly, characterization of the enzymes involved in GC metabolism has greatly expanded our understanding of GC signaling in target tissues. In man, the biologically active GC (cortisol) and the biologically inactive GC (cortisone) are interconverted by the tissue-specific expression of the type 1 and type 2 11β-hydroxysteroid dehydrogenase enzymes (HSD1 and HSD2). Much of the research on GC metabolism in peripheral target tissues has focused on the role of HSD1 in amplifying the effects of GCs in liver and adipose tissue or on the role of HSD2 in blocking the effects of GCs in the kidney and placenta. In contrast, the role of GC metabolism in modulating the effects of GCs on fetal lung maturation and the pulmonary surfactant system in humans is less understood. The goal of this review article is to present a brief overview of the role of GCs in human fetal lung maturation and pulmonary surfactant production, and to familiarize the reader with the biochemistry of the metabolism of natural and synthetic GCs by the HSD enzymes. In addition, we will review data concerning the expression and activity of the HSD enzymes in the human fetal lung and contrast this to what is known about the HSD enzymes in the fetal rodent lung. Although rodents, rabbits, sheep, and several primates have been invaluable model systems for the study of fetal lung development, we have chosen to largely focus this review on human lung, since there are significant differences in GC metabolism between humans and other species.

Surfactant protein A in rabbit sinus and middle ear mucosa

In the present study, pulmonary surfactant protein A (SP-A) messenger RNA (mRNA) and protein were characterized in adult rabbit middle ear and maxillary sinus. Fifteen adult rabbits were used for the study: 6 with evidence of acute middle ear infections and maxillary sinusitis, 6 with infections that were successfully treated with tetracycline, and 3 that were pathogen-free. We detected SP-A mRNA in maxillary sinus and middle ear tissues by Northern blot analysis and reverse transcriptase—polymerase chain reaction (RT-PCR). The RT-PCR also revealed the presence of SP-B and SP-C mRNA in middle ear and sinus tissues. We detected SP-A protein, of molecular weight approximately 29 and 70 kd, in middle ear and sinus tissues by immunoblot analysis. Unlike the SP-A protein present in the lung, the molecular weight of the SP-A protein present in the middle ear and paranasal sinus was not altered by digestion with an enzyme that cleaves N-linked carbohydrates. Immunostaining and in situ hybridization showed that SP-A protein and mRNA, respectively, were present in surface epithelial cells of the middle ear and in epithelial cells of submucosal glands in sinus tissues. These data provide the first evidence of the presence of pulmonary surfactant proteins in the paranasal sinuses and confirm previous reports of SP-A in the middle ear epithelium.

Characterization of rabbit surfactant-associated proteins

In the present study, the apolipoproteins associated with a purified surfactant fraction isolated from lung lavage of adult rabbits were characterized. Surfactant purity was assessed by the glycerophospholipid composition and by electron microscopic examination. The purified surfactant was delipidated and the apolipoproteins were analyzed by two-dimensional polyacrylamide gel electrophoresis. By use of this technique at least eight proteins or families of proteins were found to be associated with surfactant. Four of these apolipoproteins were families of proteins of 55-70, 29-36, 26-28 and 22-23 kilodaltons (kDa). All of these apolipoprotein families had acidic isoelectric points (pI less than or equal to 5.6), and were specifically bound to a Con A-Sepharose matrix, indicative that these apolipoprotein families are modified by oligosaccharide side-chains. The finding that neuraminidase treatment degraded the 29-36 kDa family is suggestive that this apolipoprotein family contains sialic acid residues. Three major proteins of 66, 43-45 and 35 kDa and a minor protein of 86 kDa were also observed. These proteins had isoelectric points in the more neutral range (pI 6.0-6.5). The 66 kDa protein (pI 6.4) had the same apparent molecular weight and isoelectric point as the major protein of delipidated rabbit serum and as purified rabbit albumin, which suggests that this protein is albumin. These findings are indicative that the apolipoproteins of surfactant are more numerous and complex than previously reported.