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J H Fisher

Publications and source records attributed to J H Fisher.

At least 19 recordsLinked to original sources
PubMed1992

Primary structure of rat pulmonary surfactant protein D. cDNA and deduced amino acid sequence.

Surfactant protein D (SP-D) is a carbohydrate-binding glycoprotein containing a collagen-like domain that is synthesized by alveolar type II epithelial cells. The complete primary structure of rat SP-D has been determined by sequencing of a cloned cDNA. The protein consists of three regions: an NH2-terminal segment of 25 amino acids, a collagen-like domain consisting of 59 Gly-X-Y repeats, and a COOH-terminal carbohydrate recognition domain of 153 amino acids. There are 6 cysteine residues present in rat SP-D: 2 in the NH2-terminal noncollagenous segment and 4 in the COOH-terminal carbohydrate-binding domain. The collagenous domain contains one possible N-glycosylation site. The protein is preceded by a cleaved, NH2-terminal signal peptide. SP-D shares considerable homology with the C-type mammalian lectins. Hybridization analysis demonstrates that rat SP-D is encoded by a 1.3-kilobase mRNA which is abundant in lung and highly enriched in alveolar type II cells. Extensive homology exists between rat SP-D and bovine conglutinin.

Amino Acid Sequence↗
PubMed1992

Multiple molecular alterations in mouse lung tumors.

Twenty-five mouse lung tumors induced by a single urethan treatment in female A/J, BALB/c, and (A/J x C3H/He)F1 (AC3) mice were analyzed for the presence of mutations at codon 61 of the Ki-ras gene and for the expression of the surfactant protein A (SP-A), retinoblastoma (Rb), growth arrest-specific-3 (gas-3), p53, c-myc, and thymidylate synthase (TS) genes. Ki-ras codon 61 mutations were detected in 22 of 25 tumor samples without differences among strains. In comparison with normal lungs, all the tumors showed increased SP-A mRNA levels, indicating their derivation from alveolar type II pneumocytes or Clara cells. Rb and gas-3 transcripts were instead found in all tumors at about tenfold and about 20-fold reduced levels, respectively. No apparent structural alterations or loss of heterozygosity at the Rb locus was detected in any tumors. The p53 mRNA was observed without variation in quantity or size in lung tumors and normal tissue. A threefold to fivefold c-myc overexpression was observed, without amplification of the gene. TS expression was only slightly increased, indicating no great differences in cell proliferation between lung tumors and normal tissue. Our data suggest that the pathogenesis of urethan-induced lung tumors in mice involves specific and recurrent molecular alterations (Ki-ras mutations, decrease of Rb and gas-3 expression, and increase of c-myc expression) that could represent different steps in lung carcinogenesis.

Animals↗
PubMed1992

Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension.

The role of endogenous circulating or locally produced endothelin-1 (ET-1) in pulmonary hypertensive states is unknown. To investigate this we measured ET-1 levels and preproendothelin-1 (prepro-ET-1) mRNA expression at various ages in control Sprague-Dawley (SDR) rats and in fawn-hooded rats (FHR), a strain which develops idiopathic pulmonary hypertension. Although serum ET-1 levels were similar in SDR and FHR, we found twofold increases in FHR whole lung homogenate ET-1 levels by radioimmunoassay. Coexisting threefold increases in preproET-1 mRNA expression were found in FHR lungs by densitometric analysis of Northern blots and by filter hybridization, suggesting the increase in lung ET-1 was due to enhanced intrapulmonary production of the peptide. To test whether the increase in lung preproET-1 mRNA was primary or secondary to established pulmonary hypertension, we compared preproET-1 mRNA expression prior to development of pulmonary hypertension in fetal (19 day gestation) and neonatal (5 day old) FHR and SDR. Despite similar right ventricular size in SDR and FHR, preproET-1 mRNA was already elevated in neonatal FHR lungs. Furthermore, we found no increase in lung preproET-1 mRNA or ET-1 levels in adult SDR with an equivalent degree of pulmonary hypertension due to chronic hypoxia, implying that the increases in ET-1 production in FHR were not a common consequence of all pulmonary hypertensive states. The functional significance of these observations remains unclear but raises the possibility of a role for ET-1 in the pathophysiology of pulmonary hypertension in the FHR.

Animals↗
PubMed1992

An antibody with specificity for surfactant protein C precursors: identification of pro-SP-C in rat lung.

Surfactant protein C (SP-C) is a lung-specific, hydrophobic peptide found in organic extracts of pulmonary surfactant. Alveolar SP-C (3.5 kD) is produced from proteolytic cleavage of a larger precursor molecule (pro-SP-C; 21 kD). While SP-C is synthesized by type II cells, the pathways for processing and secretion have remained elusive due, in part, to the lack of monospecific antibodies against SP-C or its precursors. This report describes production and characterization of a new antibody directed against pro-SP-C epitopes. Polyclonal antisera (anti-CPRO-SP-C) was prepared using a synthetic peptide corresponding to a portion of rat SP-C cDNA sequence (Ile26-Ser72). This contained amino acids 3-35 of mature SP-C plus additional C-terminal residues (His59-Ser72). On Western blots, anti-CPRO-SP-C competitively reacted to CPRO-SP-C but not to mature SP-C. Immunoblots of in vitro synthesized pro-SP-C confirmed that the antisera also recognized native protein. Immunocytochemistry with anti-CPRO-SP-C demonstrated staining for pro-SP-C peptides in isolated type II cells as well as in alveolar epithelial cells of rat lung sections. Pro-SP-C preferentially co-localized to cells that stained positive for Maclura pomifera antigen. Anti-CPRO-SP-C staining was not observed in lung interstitium, pulmonary vasculature, or several control tissues (brain, heart, and liver were negative). Western blotting of subcellular fractions demonstrated pro-SP-C peptides in plasma membrane (20 kD) and microsomal (20 and 21 kD) fractions with a 16 kD peptide present in lamellar bodies. No pro-SP-C peptides were detected in purified surfactant. These results demonstrate the use of a synthetic peptide to generate specific antiserum against more hydrophilic domains of pro-SP-C sequences and confirm that SP-C propeptides are unique to the lung.

Amino Acid Sequence↗
PubMed1991

In vivo regulation of surfactant proteins by glucocorticoids.

Surfactant proteins have key roles in regulating surfactant secretion, in recycling, and in the assembly of the surfactant monolayer but little is known about their regulation in vivo. Surfactant proteins SP-A, SP-B, and SP-C have been shown to be upregulated by glucocorticoids in vitro, but the role of glucocorticoids in the physiologic regulation of surfactant protein synthesis remains unknown. We have studied the effects of exogenously administered glucocorticoids on the regulation of steady-state surfactant protein mRNA accumulation. We have also studied the effects of adrenalectomy on the accumulation of the surfactant protein mRNAs. Surfactant protein genes appear to have quantitatively different responses to exogenously administered glucocorticoids, with SP-C mRNA increasing at the lowest dose, SP-A and SP-B mRNA increasing in response to similar glucocorticoids doses but with SP-B yielding the highest maximum response. Adrenalectomy, however, does not alter surfactant protein mRNA levels. These observations support a minor role for glucocorticoids in maintaining the steady-state accumulation of surfactant protein mRNA. Adrenalectomy decreases total pulmonary SP-A when compared to sham-operated animals in the absence of changes in its mRNA. Therefore, glucocorticoids may have translational or post-translational effects that regulate total pulmonary SP-A accumulation, but the effects appear to be minor. These findings support a potential role for the adrenal in the pulmonary response to stress and demonstrate for the first time differential accumulation of the surfactant protein mRNAs to glucocorticoids in vivo.

Adrenalectomy↗
PubMed1990

Expression and characterization of rat surfactant protein A synthesized in Chinese hamster ovary cells.

Rat surfactant protein A (SP-A) was expressed in a Chinese hamster ovary (CHO-K1) cell line and characterized for biologic activity using assays for receptor binding and modulation of phospholipid secretion from isolated type II cells. The CHO-K1 cell line was cotransfected with separate plasmids encoding for the rat SP-A, dihydrofolate reductase and neomycin phosphotransferase, respectively. Antibiotic (Geneticin-G418)-resistant transformants were screened by ELISA for the secretion of recombinant SP-A into the media. Northern analysis of the transfected cell lines demonstrated the expression of both 1.6 kb and 0.9 kb mRNA species for SP-A, consistent with the proposed differential polyadenylation of the primary transcript. Amplification with methotrexate resulted in a dose-dependent increase in mRNA for SP-A and a 20-fold increase in the production of recombinant SP-A relative to untreated cells. Maximum production of SP-A was 370 micrograms of SP-A/l of media in a 4-day incubation. Recombinant SP-A was purified from the serum-free media of large scale cultures of transfected, amplified CHO cells by affinity chromatography on mannose-Sepharose. The recombinant SP-A migrated similarly to native SP-A by NaDodSO4-PAGE analysis under reducing and nonreducing conditions and under reducing conditions after digestion with N-glycanase. Recombinant SP-A effectively competed with 125I-native SP-A for binding to the high affinity receptor for SP-A on isolated plasma membranes from rat alveolar type II cells. The recombinant SP-A was as effective as native SP-A in the inhibition of secretion of phospholipid from isolated type II cells. We conclude that recombinant rat SP-A produced in Chinese hamster ovary cells is physically and functionally similar to native rat SP-A.

Animals↗
PubMed1990

Human aminoacylase-1: cloning, regional assignment to distal chromosome 3p21.1, and identification of a cross-hybridizing sequence on chromosome 18.

Aminoacylase-1 (ACY1, EC 3.5.1.14) is a cytosolic enzyme with a wide range of tissue expression and has been postulated to function in the catabolism and salvage of acylated amino acids. ACY1 has been assigned to chromosome 3p21, a region reduced to homozygosity in small-cell lung cancer and renal cell carcinoma, and has been reported to exhibit reduced or absent expression in small-cell lung cancer cell lines and tumors. Using monoclonal antibodies to human ACY1, we have isolated cDNA clones from a liver lambda gt11 cDNA library. As proof of identity, the fusion protein encoded by a putative ACY1 cDNA displayed ACY1 enzymatic activity. Additionally, it was determined that the putative ACY1 cDNA clones hybridize to an EcoR1 restriction fragment that has been mapped to chromosome 3p. Both ACY1 activity and this restriction fragment have been further demonstrated to be syntenic to distal 3p21.1 through the use of a panel of human-rodent somatic cell hybrids containing fragments of chromosome 3. An additional EcoR1 restriction fragment to which the probe hybridizes has been assigned to chromosome 18. The major mRNA species to which the ACY1 cDNA hybridizes is 0.9 kb; faint hybridization to a 4.2-kb mRNA species is also detected. These studies further refine a region of interest in the investigation of gene inactivation in small-cell lung cancer and provide a new marker on chromosome 18.

Amidohydrolases↗
PubMed1990

Alpha-skeletal actin messenger RNA increases in acute right ventricular hypertrophy.

Newborn calves exposed to hypobaric hypoxia develop severe pulmonary hypertension. Right ventricular hypertrophy and failure occur as a consequence of the increased pressure load. Alterations in right ventricular myocyte performance or differentiation could be reflected by the changes in the expression of contractile protein genes. We studied expression of contractile actin isotypes by measuring mRNA levels in total cellular RNA purified from right (RV) and left ventricles (LV) of calves with severe pulmonary hypertension after a 2-wk exposure to hypobaric hypoxia and age-matched controls. alpha-Skeletal actin mRNA was increased greater than 10-fold in the RV of hypertensive animals, whereas alpha-cardiac actin mRNA did not appear to change. alpha-Skeletal actin mRNA and alpha-cardiac actin mRNA did not increase in the LV of any of the hypoxic animals. After a 2-wk hypoxic exposure, calves were removed from the chamber. Two days later, RV alpha-skeletal actin mRNA decreased dramatically but was apparently elevated above that of an age-matched control. Thirty days after hypoxia, there appeared to be a persistent increase in RV alpha-skeletal actin mRNA. Although the physiological significance of these changes are unknown, an alteration in the RV myocyte phenotype has occurred.

Actins↗
PubMed1990

Effect of a reconstituted basement membrane on expression of surfactant apoproteins in cultured adult rat alveolar type II cells.

Pulmonary surfactant, which is composed of phospholipids and three lung-specific apoproteins, is synthesized and secreted by alveolar type II cells. Previous work from this laboratory (Biochim. Biophys. Acta 1987; 931:143-156) has shown that cell-extracellular matrix interactions and cuboidal cell shape affect both the ultrastructural appearance and pattern of phospholipids synthesized by cultured rat type II cells. In the present study, we have examined the effects of cell-matrix interactions and cell shape on the ability of adult rat type II cells to express the surfactant apoproteins in culture. Isolated adult rat type II cells were cultured for 2, 4, and 8 days on either tissue culture plastic, on an extract of the Engelbreth-Holm-Swarm (EHS) tumor, or on laminin-coated plastic dishes. Expression of surfactant proteins A, B, and C (SP-A, SP-B, and SP-C) was evaluated by Northern analysis using specific rat cDNA probes for these mRNAs. SP-A content was determined by enzyme-linked immunosorbent assay using a polyclonal antibody raised against rat SP-A purified from lavage. Type II cells cultured on plastic dishes assumed an attenuated morphology soon after being placed in culture. Except for an occasional positive signal on day 2 of culture, these cells were uniformly negative for the presence of mRNA for SP-A, SP-B, or SP-C. Type II cells cultured on plastic did not contain SP-A. In contrast, type II cells cultured on EHS gels formed three-dimensional aggregates on the surface of the substratum; these aggregates were composed of polarized cells that had their apical surfaces directed inward. Type II cells cultured on this substratum showed a positive signal for mRNA for all three surfactant proteins; the abundance of these mRNAs, however, was significantly below that seen in freshly isolated type II cells. While the abundance of mRNA for SP-A and SP-B steadily increased with time in culture under these conditions, the abundance of SP-C mRNA decreased, suggesting that SP-C is regulated independently of SP-A and SP-B. These cultures were also positive for SP-A content, which increased with increasing time in culture. Type II cells cultured on laminin-coated dishes initially spread more slowly across the culture surface than cells on plastic, but were extremely attenuated by day 8 in culture. These cells contained neither SP-A, nor mRNA for any of the three surfactant proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1989

Nucleotide and deduced amino acid sequence of the hydrophobic surfactant protein SP-C from rat: expression in alveolar type II cells and homology with SP-C from other species.

Pulmonary surfactant lowers surface tension in the lung. Its deficiency leads to the severe physiologic abnormalities seen in the respiratory distress syndrome. The hydrophobic surfactant proteins, SP-B and SP-C, appear to be especially important in the surface-spreading characteristics of pulmonary surfactant. We report the nucleotide sequence of cDNA clones for rat SP-C and compare the deduced amino acid sequence for SP-C from several species. A highly conserved domain exists within the confines of mature human SP-C. An Eisenberg plot of this region predicts a membrane-associated helix. We also demonstrate by Northern analysis the tissue-specific expression of SP-C. A comparison of signal strength between total lung RNA and RNA derived from isolated type II cells supports the idea that most SP-C messenger RNA in total lung can be accounted for by that present in alveolar type II cells.

Amino Acid Sequence↗
PubMed1989

cDNA and deduced amino acid sequence for the rat hydrophobic pulmonary surfactant-associated protein, SP-B.

Pulmonary surfactant prevents collapse of lung alveoli by lowering surface tension at the air/liquid interface. The hydrophobic surfactant associated proteins SP-B and SP-C have been shown to be important in surfactant function and metabolism. A cDNA clone for rat SP-B was isolated and sequenced. Northern analysis showed mRNA for SP-B was present in whole lung and was greatly enriched in alveolar type II cells, but was not present in brain, kidney, spleen or liver. A full length transcript of the rat SP-B cDNA clone consists of 1536 bases and encodes an open reading frame of 376 amino acids. The predicted molecular mass of the primary translation product is 42 kDa and the predicted molecular mass of the mature protein is 8 kDa. Extensive homology exists between the rat sequence for SP-B and those reported for human and canine SP-B. The position of 25 cysteine residues has been extremely well preserved across all three species. An N-linked glycosylation site in the COOH region has been conserved across all three species. A search of the NIH database revealed homology between rat SP-B and the active site for the mouse contrapsin serum proteinase inhibitor.

Amino Acid Sequence↗
PubMed1989

Esophageal adenocarcinoma 20 years after esophageal atresia repair.

We report a case of esophageal adenocarcinoma 20 years after esophageal atresia repair. From one case report it is premature to recommend cancer surveillance for all esophageal atresia patients. However, the first survivors are now reaching an age when esophageal cancer related to chronic esophagitis may become more prevalent.

Adenocarcinoma↗
PubMed1989

Ontogeny of surfactant apoproteins in the rat.

Content of the 26-38-kD surfactant apoprotein (SP-A) was determined in lung homogenates from fetal (17-21 d gestation), postnatal (1-28 d of life), and adult male and female rats by a double sandwich ELISA. Expression of mRNA for SP-A as well as the hydrophobic apoproteins, SP-B and SP-C, were also determined in lung homogenates from fetal and adult rats of both sexes by Northern blot analysis. SP-A was undetectable in fetal lungs on d 17 (day of birth = d 22) and barely detectable on d 18. On d 19 there was a 3- to 4-fold increase in SP-A content above d 18 levels. Between d 19 and 21 SP-A content significantly increased another 6- to 9-fold. SP-A content on the day of birth was not significantly different from that seen on gestational d 21. SP-A content decreased 35-40% between the day of birth and postnatal d 7. After the second postnatal week SP-A content gradually increased, reaching adult levels after d 28. No sex differences in SP-A content were observed during fetal or postnatal lung maturation. SP-A mRNA was first detected in fetal lungs on d 18 and increased in relative abundance until d 21, but remained below adult levels. Developmental changes in fetal lung SP-A content closely paralleled changes in fetal expression of SP-A mRNA. SP-B mRNA was also first detected on d 18, then increased in relative abundance to adult levels by d 20. SP-C mRNA was clearly detectable on d 17, then increased in relative abundance to adult levels by d 20-21. Unlike surfactant phospholipids, there are no apparent sex differences in the expression of any of the surfactant apoproteins during late gestation. The differences observed during fetal lung maturation in the time of onset and changes in relative abundance among the three apoprotein mRNA imply that their genes may be differentially regulated in the developing rat lung.

Animals↗
PubMed1988

Rat pulmonary surfactant protein A is expressed as two differently sized mRNA species which arise from differential polyadenylation of one transcript.

In rat, the surfactant-associated glycoprotein A, SP-A, is encoded by two mRNA species of 0.9 and 1.6 kb. Each transcript is polyadenylated and is found in approximately the same ratio in total cellular RNA isolated from either alveolar type II cells or from whole lung. The two mRNA species have identical coding regions and differ only in the length of the 3' untranslated sequences. Restriction analysis and partial sequence analysis of rat genomic clones indicate that each mRNA species arises from one gene, with the difference in size most easily accounted for by differential polyadenylation. During fetal development, the accumulation of mRNA encoding SP-A is detectable by day 18 but increases many-fold on day 19. However, there are no apparent alterations in the prevalence of either mRNA species during fetal development or in the early postnatal period.

Amino Acid Sequence↗