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Biomedical subjects

T E Weaver

Publications and source records attributed to T E Weaver.

At least 37 records · Page 2Linked to original sources
PubMed1997

Expression of glycogen phosphorylase isozymes in developing rat lung.

Glycogen accumulates to significant levels in epithelial cells of the developing respiratory tract. Mobilization of glycogen stores is regulated differentially along the respiratory epithelium such that glycogenolysis in the alveolar epithelium (the site of surfactant synthesis) precedes that in the bronchial and bronchiolar epithelium. The initial step in glycogen degradation is catalyzed by glycogen phosphorylase, which exists as three genetically distinct isozymes referred to as muscle, liver, and brain isoforms. The goal of this study was to characterize the temporal and spatial expression of each of the glycogen phosphorylase isozymes in developing lung to determine which isoform(s) was associated with glycogen mobilization in the fetal type II epithelial cell. RNA levels encoding glycogen phosphorylase were assessed by ribonuclease protection assay using isoform-specific antisense probes. RNAs encoding the brain and liver isozymes were detected in isolated day 20 fetal type II epithelial cells and at lower levels in adult type II cells. The muscle isoform RNA was barely detectable in fetal type II cells and was undetectable in adult type II cells. Expression of brain and liver isoform RNAs was higher in whole fetal lung than in fetal type II cells. Consistent with this result, in situ hybridization studies demonstrated widespread expression of the brain and liver isoforms in developing lung tissues; in contrast, expression of the muscle isoform was restricted to the pulmonary vein. Glycogen phosphorylase enzyme activity corresponding to the brain isoform was clearly detected in isolated fetal type II cells; however, the majority of enzyme activity migrated as two bands with distinct electrophoretic mobilities that may have been the result of isoform heterodimerization. Collectively, these results suggest that the brain and liver isoforms of glycogen phosphorylase may be involved in mobilization of type II cell glycogen during late fetal lung development.

Animals↗
PubMed1997

Pulmonary dysfunction in neonatal SP-B-deficient mice.

Pulmonary function was assessed in newborn wild-type and homozygous and heterozygous surfactant protein B (SP-B)-deficient mice after birth. SP-B +/+ and SP-B+/- mice became well oxygenated and survived postnatally. Although lung compliance was decreased slightly in the SP-B+/- mice, lung volumes and compliances were decreased markedly in homozygous SP-B-/- mice. They died rapidly after birth, failing to inflate their lungs or oxygenate. SP-B proprotein was absent in the SP-B-/- mice and was reduced in the SP-B+/- mice, as assessed by Western analysis. Surfactant protein A, surfactant proprotein C, surfactant protein D, and surfactant phospholipid content in lungs from SP-B+/- and SP-B-/- mice were not altered. Lung saturated phosphatidylcholine and precursor incorporation into saturated phosphatidylcholine were not influenced by SP-B genotype. Intratracheal administration of perfluorocarbon resulted in lung expansion, oxygenation, and prolonged survival of SP-B-/- mice and in reduced lung compliance in SP-B+/+ and SP-B+/- mice. Lack of SP-B caused respiratory failure at birth, and decreased SP-B protein was associated with reduced lung compliance. These findings demonstrate the critical role of SP-B in perinatal adaptation to air breathing.

Animals↗
PubMed1997

Decreased lung compliance and air trapping in heterozygous SP-B-deficient mice.

Genetic ablation of the murine SP-B gene in transgenic mice caused lethal perinatal respiratory distress in homozygous offspring, whereas heterozygous SP-B (+/-) mice survived postnatally. In adult SP-B(+/-) mice, surfactant protein B mRNA and the alveolar lavage SP-B protein were reduced by 50% compared with wild-type littermates, consistent with the inactivation of a single SP-B allele. Expression of SP-A, SP-C, and SP-D proteins was not affected in SP-B(+/-) mice. Heterozygous SP-B(+/-) mice reached maturity in numbers expected by Mendelian inheritance of a recessive gene. Lung morphology and both intracellular and extracellular phospholipid pool size and composition were unaltered in the SP-B(+/-) mice. Despite normal survival, pulmonary function studies demonstrated a consistent decrease in lung compliance in SP-B(+/-) mice. Abnormalities of inflation/deflation curves demonstrated airway collapse at low deflation pressures. Residual volumes were increased in the SP-B(+/-) mice. In summary, SP-B mRNA and SP-B protein were reduced by 50% in SP-B(+/-) mice, resulting in abnormalities of lung compliance and air trapping, suggesting a potential susceptibility to pulmonary dysfunction associated with SP-B deficiency.

Animals↗
PubMed1997

An instrument to measure functional status outcomes for disorders of excessive sleepiness.

This article reports the development of the functional outcomes of sleep questionnaire (FOSQ). This is the first self-report measure designed to assess the impact of disorders of excessive sleepiness (DOES) on multiple activities of everyday living. Three samples were used in the development and psychometric analyses of the FOSQ: Sample 1 (n = 153) consisted of individuals seeking medical attention for a sleep problem and persons of similar age and gender having no sleep disorder; samples 2 (n = 24) and 3 (n = 51) were composed of patients from two medical centers diagnosed with obstructive sleep apnea (OSA). Factor analysis of the FOSQ yielded five factors: activity level, vigilance, intimacy and sexual relationships, general productivity, and social outcome. Internal reliability was excellent for both the subscales (alpha = 0.86 to alpha = 0.91) and the total scale (alpha = 0.95). Test-retest reliability of the FOSQ yielded coefficients ranging from r = 0.81 to r = 0.90 for the five subscales and r = 0.90 for the total measure. The FOSQ successfully discriminated between normal subjects and those seeking medical attention for a sleep problem (T157 = -5.88, p = 0.0001). This psychometric evaluation of the FOSQ demonstrated parameters acceptable for its application in research and in clinical practice to measure functional status outcomes for persons with DOES. Thus, the FOSQ can be used to determine how disorders of excessive sleepiness affect patients' abilities to conduct normal activities and the extent to which these abilities are improved by effective treatment of DOES.

Activities of Daily Living↗
PubMed1996

Structural requirements for targeting of surfactant protein B (SP-B) to secretory granules in vitro and in vivo.

Human surfactant protein B (SP-B) is synthesized by type II cells as a 381-residue preproprotein which is proteolytically processed to a 79-residue mature peptide and targeted to lamellar bodies for secretion. To identify secretory granule targeting determinants, constructs encoding the SP-B preproprotein (SP-B), COOH-terminally deleted SP-B (SP-BDeltaC), the NH2-terminal propeptide (SP-BN), and a chimeric molecule consisting of albumin and the mature peptide (ALB/SP-BM) were transfected into AtT-20 and PC12 cells. Pulse-chase studies demonstrated that 10-30% of SP-B and SP-BDeltaC remained in cells in an endoglycosidase H-resistant form. Secretion of stored SP-B was stimulated by forskolin/12-O-tetradecanoylphorbol-13-acetate and intracellular SP-B was localized to secretory granules by immunoelectron microscopy. In contrast, SP-BN and ALB/SP-BM were constitutively secreted and not detected in secretory granules. Specific processing of SP-B was not detected in either AtT-20 or PC12 cells. Expression of SP-BDeltaC in transgenic mice resulted in secretion of fully processed mature SP-B, indicating correct processing and targeting of this construct in vivo. We conclude that 1) SP-B processing occurs in a cell-specific manner, 2) the proprotein contains secretory granule targeting determinants that are not cell-specific, 3) the NH2-terminal propeptide and the mature peptide are required for targeting SP-B to lamellar body, and 4) the COOH-terminal propeptide is not required for processing or sorting of SP-B.

Animals↗
PubMed1996

Structural requirements for intracellular transport of pulmonary surfactant protein B (SP-B).

Human SP-B is synthesized by the alveolar Type II epithelial cell as a 381 amino acid preproprotein. The 79 residue mature SP-B peptide is extremely hydrophobic and flanked by propeptides of 200 and 102 amino acids at its NH2- and COOH-termini, respectively. The purpose of this study was to identify peptide domains of the SP-B proprotein necessary for trafficking of the mature peptide in the secretory pathway. To this end several constructs were generated, by subcloning the full length human SP-B (SP-B), COOH-terminally truncated SP-B (SP-B delta C, in which residues 201-381 were deleted), NH2-terminally deleted SP-B (SP-B delta N, in which residues 28-200 were deleted), NH2-terminal propeptide (SP-BN), mature SP-B (SP-BM) and COOH-terminal propeptide (SP-BC), into the mammalian expression vector pcDNA3. The resulting expression constructs were characterized by DNA sequencing and in vitro transcription/translation and subsequently transfected into Chinese hamster ovary cells. 48 h after transfection, cells were labeled with [35S]-met/cys and analyzed by immunoprecipitation, SDS-PAGE and autoradiography. Proteins encoded by SP-B, SP-B delta C, SP-BN and SP-BC constructs were secreted into media; in contrast, SP-B constructs lacking the NH2-terminal propeptide (SP-B delta N) remained in the endoplasmic reticulum (as assessed by endoglycosidase H sensitivity) and were rapidly degraded. We conclude that (1) 27 amino acids at the NH2-terminus of SP-B contain a functional signal peptide and (2) the NH2-terminal propeptide of the SP-B precursor is necessary and sufficient for intracellular trafficking of the mature peptide.

Animals↗
PubMed1996

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-gammaT) plays a critical role in regulation of glycogen mobilization in developing lung.

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-gammaT) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-gammaT RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-gammaT protein expression was temporally and spatially correlated with expression of PhK-gammaT RNA. PhK-gammaT RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-gammaT RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-gammaT protein was virtually undetectable in gsd/ gsd Type II cells and enzyme activity was very low. These results suggest that PhK-gammaT plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-gammaT RNA or unstable protein.

Amino Acid Sequence↗
PubMed1996

Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis.

Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung. To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E. coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein. Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas. Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen. Hybridomas producing high-affinity antibodies were subcloned by limiting dilution. Antibodies from tissue culture fluid from an IgG1 clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized. The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lysates of cell lines known to express TTF-1 mRNA. MAb 8G7G3/1 also stained nuclei of tissue in a highly specific manner consistent with the pattern of expression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization. MAb 8G7G3/1 was used for TTF-1 immunohistochemistry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung. TTF-1 was detected in primary lung adenocarcinomas and small cell carcinomas and was absent in colon and breast carcinomas. These findings demonstrate that anti-TTF-1 MAb 8G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can be used to distinguish between lung and nonlung origin of a tumor.

Adenocarcinoma↗
PubMed1996

Neurobehavioral consequences of arousals.

The neurobehavioral deficits of obstructive sleep apnea syndrome (OSAS) are often attributed to the rate of respiratory disturbance or rate of arousals during sleep. However, sleep disordered breathing is also associated with other changes in sleep infrastructure that may account for cumulative waking deficits. This was illustrated in polysomnographic data from 1,521 patients with OSAS where increasing arousal indices were associated with increased duration of stage 1 sleep and concomitant reduction in total sleep time. Similar results have been found in paradigms in which sleep was experimentally fragmented in healthy individuals. It appears that chronic fragmentation of sleep, whether by apneas or acoustic stimuli, leads to cumulative homeostatic pressure for sleep, which may explain a number of phenomenon characteristic of both untreated OSAS patients and experimentally fragmented sleepers: (1) increased arousal threshold, (2) rapid return to sleep after arousal, (3) fewer awakenings over time, (4) increased sleep inertia on awakenings, (5) increased amnesia for arousals, and (6) daytime sleepiness. Elevated homeostatic drive for sleep appears to be a function of both the frequency of arousals within a night and the chronicity of sleep fragmentation across nights, neither of which have been adequately modeled in experimental studies of healthy subjects.

Arousal↗
PubMed1995

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.

Animals↗
PubMed1995

Posttranslational processing of surfactant protein C in rat type II cells.

Pulmonary surfactant consists of phospholipids and proteins that form a stable monolayer at the surface of the alveoli to prevent lung collapse. Surfactant protein C (SP-C) is a hydrophobic 4-kDa palmitoylated protein derived from a 21-kDa precursor. We determined the membrane insertion, proteolytic processing, and subcellular location of 21-kDa proSP-C. In vitro, proSP-C associated with canine microsomes, and the NH2-terminal of proSP-C was protected from digestion with proteinase K, suggesting that proSP-C was inserted in a type III transmembrane configuration. Treatment of freshly isolated rat type II cells with cerulenin blocked acylation of the 21-kDa precursor. Pulse-chase labeling of type II cells demonstrated proSP-C processing intermediates of 19, 16, and 13 kDa that contained the NH2-terminal of proSP-C. Proteolytic processing of proSP-C was inhibited by incubation at 20 degrees C, suggesting that processing of proSP-C begins in a late Golgi or post-Golgi compartment. Immunogold labeling of rat lung with an antiserum to the NH2-terminal of proSP-C identified proSP-C in the trans-Golgi and multivesicular bodies but not in lamellar bodies. These findings suggest that proSP-C processing takes place in the trans-Golgi and multivesicular bodies before SP-C is incorporated into lamellar bodies.

Animals↗
PubMed1995

Human surfactant protein B: structure, function, regulation, and genetic disease.

Elucidation of the structure and function of the hydrophobic surfactant protein (SP-B) and the SP-B gene has provided critical insight into surfactant homeostasis and control of respiratory epithelial cell gene expression. Surfactant protein B, in concert with surfactant protein A (SP-A), surfactant protein C (SP-C), and surfactant phospholipids, contributes to the structure and function of surfactant particles, determining surface activities and pathways by which surfactant phospholipids and proteins are processed, routed, packaged, and secreted from lamellar bodies by type II epithelial cells. After secretion, SP-B plays an essential role in determining the structure of tubular myelin, the stability and rapidity of spreading, and the recycling of surfactant phospholipids. The biochemical and structural signals underlying the homeostasis of alveolar surfactant are likely mediated by interactions between the surfactant proteins and phospholipids producing discrete structural forms that vary in size, aproprotein, and phospholipid content. Distinctions in structure, protein, and size are likely to determine the function of surfactant particles, their catabolism, or recycling by alveolar macrophages and airway epithelial cells. Analysis of the genetic controls governing the SP-B gene has led to the definition of DNA-protein interactions that determine respiratory epithelial cell gene expression in general. The important role of SP-B in lung function was defined by the study of a lethal neonatal respiratory disease, hereditary SP-B deficiency, caused by mutations in the human SP-B gene.

Disease Models, Animal↗
PubMed1995

Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues.

Replication-deficient adenoviruses have been used to transfer various genes of interest to mammalian tissues in vivo. Effective gene therapy for inborn genetic defects presenting with significant morbidity and mortality at birth will require correction of the defect prenatally. To test the hypothesis that intra-amniotically administered adenovirus transfers gene expression to fetal tissues, replication-deficient human type 5 adenovirus carrying the lacZ gene which encodes nuclear-targeted bacterial beta-galactosidase (Av1LacZ4) was instilled into the amniotic cavity of fetal sheep (10(10) to 1.5 x 10(11) pfu) and fetal mice (10(9) pfu) at 0.8 term gestation. Amniotic membranes and gastrointestinal and respiratory tract tissues were harvested after 3 d, bacterial beta-galactosidase activity was determined by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-gal) enzyme-histochemistry, and tissue integrity was assessed in sections stained with hematoxylin and eosin. Bacterial beta-galactosidase activity was abundant in amniotic membranes and present in lower levels in esophagus, stomach, and small intestine as well as in conducting airways and pulmonary alveoli. To determine whether gene transfer by intraamniotic injection of adenovirus was dose-dependent, Av1Luc1, an adenoviral vector carrying the gene for luciferase (10(5)-10(9) pfu), was injected intraamniotically into fetal mice at 0.8 term gestation. Luciferase activity measured after 3 d in tissue homogenates of Av1Luc1-treated fetal mice revealed a linear dose response in amniotic membranes and gastrointestinal and respiratory tract organs. Intraamniotic administration of an adenoviral gene vector leads to expression of the transferred gene in amniotic membranes as well as in fetal gastrointestinal and respiratory tract tissues in a dose-dependent manner.

Adenoviruses, Human↗
PubMed1994

Characterization of a rat cDNA clone encoding calcium/calmodulin-dependent protein kinase I.

Two cDNA clones encoding calcium/calmodulin-dependent (CaM) protein kinase I were isolated. In contrast to the previously reported CaM kinase I cDNA, which encodes a protein with a mass of 37 kDa, the clones identified in this study encode a protein (10-1/CaM kinase I) with a predicted mass of 42 kDa; the size of 10-1/CaM kinase I was verified by hybrid-selected translation.

Amino Acid Sequence↗
PubMed1994

Adenoviral-mediated gene transfer of human surfactant protein B to respiratory epithelial cells.

Human surfactant protein B (SP-B) is a 79-amino acid, phospholipid-associated polypeptide expressed by respiratory epithelial cells of the lung. SP-B is essential for lung function, enhancing the spreading and stability of surfactant phospholipids that serve to reduce surface tension at the alveolar air-liquid interface. Congenital absence of SP-B results in neonatal respiratory failure and death. In the present work, we constructed a replication-deficient adenoviral vector, Av1SP-B1, in which the human SP-B cDNA is expressed under control of the Rous sarcoma virus (RSV) promoter in an E1-E3-deleted adenovirus type 5 (Ad5)-based vector system. Av1SP-B1 was produced in 293 kidney cells, directing the synthesis of the SP-B protein and SP-B peptides. Av1SP-B1 directed the synthesis of SP-B mRNA, precursor and active 8-9 kD polypeptide in immortalized mouse lung epithelial cells (MLE-12 cells), demonstrating complete processing to the human SP-B protein by these cells. Synthesis of human SP-B mRNA was detected as early as 12 h after infection and was maximal 48 h after infection in vitro. Northern blot analysis demonstrated that human SP-B mRNA was expressed in the lungs of cotton rats infected with Av1SP-B1 but not in those of uninfected animals or in animals infected with a reporter adenoviral vector, Av1LacZ4. In situ hybridization demonstrated the abundance and localization of the transferred human SP-B mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1993

Biosynthetic routing of pulmonary surfactant proteins in alveolar type II cells.

Surfactant proteins A, B, and C (SP-A, SP-B, and SP-C) are synthesized in alveolar type II cells. SP-B and SP-C are both synthesized as large precursor molecules that are proteolytically processed to their mature sizes. In a previous immunoelectron microscopic study, we showed that precursor SP-B is processed to its mature size in multivesicular bodies. In the present study, using a specific antibody against precursor SP-C, we demonstrate that precursor SP-C is present in the same intracellular compartments of the biosynthetic pathway, i.e., endoplasmic reticulum, Golgi complex, and multivesicular bodies, as precursor SP-B. Since mature SP-C is known to be present in multilamellar bodies, this suggests a biosynthetic routing and site of processing of this protein similar to those of SP-B. Double-labeling experiments using antibodies against SP-A, precursor SP-B, precursor SP-C, and an antibody against HA I, an adaptor protein involved in the budding of transport vesicles from the Golgi complex, showed that the different surfactant proteins traverse and exit the Golgi complex via the same route. The surfactant proteins do not exit the Golgi complex via HA I-positive coated buds or vesicles. These data are in accordance with the concept that SP-A, SP-B, and SP-C are transported together through the same biosynthetic pathway via multivesicular bodies to multilamellar bodies.

Antibodies, Monoclonal↗
PubMed1993

Cloning of the rat cyclin-dependent kinase 4 cDNA: implication in proliferation-dependent expression in rat tissues.

To identify protein kinases that may regulate fetal growth and differentiation, we used an oligonucleotide probe encoding the conserved sequence of serine/threonine kinases to screen a fetal lung cDNA library. Several clones were isolated and sequenced, one of which encodes the rat homolog of the 34 kilodalton cyclin-dependent kinase 4 (p34cdk4). Northern blot analyses of pre- and postnatal rat tissues show that rat cdk4 is expressed in a developmentally regulated pattern in all tissues examined. The mRNA is also significantly decreased in cells that are arrested in the G1 phase of cell cycle. The regulated expression of rat cdk4 is consistent with a proliferation-, rather than a differentiation-, dependent pattern.

Age Factors↗