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D Phelps

Publications and source records attributed to D Phelps.

17 recordsLinked to original sources
PubMed1996

Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts.

Human diploid fibroblasts (HDFs) can be grown in culture for a finite number of population doublings before they cease proliferation and enter a growth-arrest state termed replicative senescence. The retinoblastoma gene product, Rb, expressed in these cells is hypophosphorylated. To determine a possible mechanism by which senescent human fibroblasts maintain a hypophosphorylated Rb, we examined the expression levels and interaction of the Rb kinases, CDK4 and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in senescent HDFs. Cellular p21 protein expression increased dramatically during the final two to three passages when the majority of cells lost their growth potential and neared senescence but p21 levels declined in senescent HDFs. During this period, p16 mRNA and cellular protein levels gradually rose with the protein levels in senescent HDFs reaching nearly 40-fold higher than early passage cells. In senescent HDFs, p16 was shown to be complexed to both CDK4 and CDK6. Immunodepletion analysis of p21 and p16 from the senescent cell extracts revealed that p16 is the major CDK inhibitor for both CDK4 and CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated proteins from radiolabeled extracts from senescent HDFs showed no other CDK inhibitors. Based upon these results, we propose that senescence is a multistep process requiring the expression of both p21 and p16. p16 up-regulation is a key event in the terminal stages of growth arrest in senescence, which may explain why p16 but not p21 is commonly mutated in immortal cells and human tumors.

Carrier Proteins↗
PubMed1989

High-level expression, purification, and characterization of recombinant human tumor necrosis factor synthesized in the methylotrophic yeast Pichia pastoris.

Human tumor necrosis factor (TNF) alpha/cachectin was expressed in the methylotrophic yeast Pichia pastoris at high levels (greater than 30% of the soluble protein) by placing the TNF cDNA under the control of regulatory sequences derived from the alcohol oxidase gene. Batch fermentor cultures at cell densities of 50 and 85 g dry cell weight/L contained approximately 6 X 10(10) and 10(11) units/L TNF bioactivity (6 and 10 g/L TNF), respectively. TNF productivity of 0.108 g L-1 h-1 was obtained in the continuous mode on glycerol- and methanol-mixed feed at 25 g dry cell weight/L cell density. TNF contained in the yeast cell lysate was soluble, displayed full cytotoxic activity, and was recognized by antibodies prepared against TNF derived from Escherichia coli. TNF was purified to greater than 95% purity with greater than 75% recovery by using three sequential chromatographic steps with a coordinated effluent-affluent buffer scheme which allowed one eluate to also serve as the loading buffer for the succeeding column. The amino acid composition, NH2-terminal amino acid sequence, isoelectric point, and minimal molecular weight determined for TNF corroborated those properties predicted from the nucleotide sequence. Sedimentation data indicated that TNF in the native form is a compact trimer held by noncovalent interactions. Circular dichroic spectra of TNF resemble those of proteins with high beta structure. TNF exhibited cachectic activity on mouse 3T3-L1 cells at about the same equivalence as the cytotoxic activity toward mouse L929 cells. In the criteria examined, TNF derived from P. pastoris closely resembles TNF derived from recombinant E. coli and human HL-60 cells.

Animals↗
PubMed1986

Isolation and characterization of cDNA clones for the 35-kDa pulmonary surfactant-associated protein.

A group of 35,000-dalton sialoglycoproteins is the major non-serum protein component of pulmonary surfactant. Tryptic fragments of these proteins were sequenced, and oligonucleotide probes were synthesized based on the amino acid sequences. A human lung cDNA library was then screened using the oligonucleotide probes, and clones coding for these proteins were identified and characterized. By in vitro transcription-translation experiments we have associated individual clones with particular proteins. The data suggest that co-translational modifications of two primary translation products account for many of the isoforms observed by two-dimensional gel electrophoresis in the precursors of 35,000-dalton sialoglycoproteins.

Amino Acid Sequence↗
PubMed1986

Characterization of bovine surfactant for infants with respiratory distress syndrome.

Exogenous surfactant treatment of surfactant-deficient disease states is now under study in a number of centers, using a variety of surfactant preparations. We have chosen one preparation because of its current and potential clinical usefulness, and we have characterized it using selected tests and assays that we thought would be necessary (although not necessarily sufficient) to justify extended clinical use. We found its lipid composition to resemble that of other surfactants derived from lung mince. There is little variation among several batches with regard to lipid composition or surface tension-lowering capability. Morphologic heterogeneity occurs in individual samples of pelleted material studied by electron microscopy. Arterial oxygenation is improved when the material is administered to animals depleted of surfactant. A low molecular weight protein was identified that reacted with antibody that specifically binds nonserum surfactant proteins in a number of animal species (including human and cow). The characteristics of this surfactant preparation should be useful for comparison as newer and simpler products become available.

Animals↗
PubMed1984

Localization of pulmonary surfactant protein during mouse lung development.

During lung development type II alveolar epithelial cells produce extracellular pulmonary surfactant. Polyclonal antibodies were produced against nonserum proteins associated with human surfactant. The present studies were designed (i) to determine if mouse surfactant proteins were antigenically cross-reactive with polyclonal antibodies directed against human surfactant proteins; and (ii) to determine surfactant protein localization during fetal, neonatal, and adult mouse lung development. Two-dimensional gel electrophoresis studies in conjunction with immunologic techniques provided evidence that mouse and human surfactant proteins shared antigenic determinants. The major monomeric form of mouse surfactant protein in a glycoprotein of approximately Mr 35,000 under reducing conditions. A less abundant form was identified as a Mr 45,000 polypeptide. Immunohistochemical localization showed that type II cells contain surfactant protein at Theiler stage 26. A gradient of immunostaining was localized within alveolar surfaces. The antigen was not detected in heart, blood vessels, or pulmonary interstitial cells. Surfactant protein was detected lining alveolar surfaces in mature adult lung. The distribution of this protein during fetal and neonatal lung morphogenesis suggests that this extracellular constituent of pulmonary surfactant may be extremely useful as a phenotypic marker with which to evaluate normal and abnormal lung development.

Animals↗
PubMed1981

Inadvertent intra-arterial injection of phentaramine: a complication of drug abuse.

A female drug abuser inadvertently injected an intravenous injection of an oral anorexiant, phentaramine (Preludin), into her own brachial artery. Severe hand pain, vasospasm, and ischemia occurred. Treatment with regional axillary block, intravenous dextran, intra-arterial priscoline, and salicylate anticoagulation may have influenced a favorable outcome. Intimal damage, vasospasm, and various additive effects after intra-arterial injection contribute to the potential for serious sequelae. Prompt diagnosis, vasodilatation, elevation and compression, pain relief and, in some cases, surgery appear to improve salvage of what is frequently a major extremity injury.

Adult↗
PubMed1980

Visualization of cellular aggregates cultured on a three dimensional collagen sponge matrix.

A one-step vital stain is described for the macroscopic visualization of histotypic cell aggregates in fetal rat lung organotypic cultures. Organotypic cultures are incubated in 0.05%-0.1% 2,3,5'-triphenyl tetrazolium chloride (TTC) in culture medium (37 degrees C). Living cells reduce the tetrazole to a water-insoluble red colored formazan. Cell aggregates appear as densely stained foci against the lighter background of the Gelfoam substrate. Stained cultures may be scanned macroscopically to determine the degree of reaggregation and assess cell viability. Identification of aggregates by TTC staining improves the efficiency of tissue processing for electron microscopy and does not alter the ultrastructural appearance of the cultured cells.

Animals↗
PubMed1979

Prophylactic cesarean section antibiotics: maternal and neonatal morbidity before or after cord clamping.

In an ongoing prospective study at 2 hospitals, 114 cesarean section patients were studied to determine whether giving ampicillin after umbilical cord clamping is as effective as perioperative ampicillin in reducing maternal postoperative morbidity. The same patients have also been studied to determine the effect of prophylactic cesarean section ampicillin on the newborn. Results have shown that prophylactic ampicillin initiated after cord clamping is as effective in reducing maternal morbidity as ampicillin initiated prior to the surgery. There is also no evidence from our study that the transplacental passage of prophylactic ampicillin increases immediate or delayed neonatal infections.

Ampicillin↗
PubMed1976

An interdisciplinary team approach--a case report.

A case study is presented as a means of demonstrating the advantage of an interdisciplinary approach. The paper familiarizes the reader with the contributions to be made by a team composed of an optometrist, speech pathologist/audiologist, psychologist, and educational diagnostician. Based on the authors' experiences, it would appear that the total team approach is a most beneficial way of assisting the child with a learning disability.

Child↗
PubMed1974

Spontaneous and amino acid-stimulated glucagon secretion in the immediate postnatal period. Relation to glucose and insulin.

The extent and significance of spontaneous glucagon secretion in the immediate postnatal period were investigated in groups of normal infants studied cross-sectionally and longitudinally. Arginine-and alanine-stimulated glucagon secretion was also studied. Plasma glucagon concentrations were correlated with prevailing glucose and insulin concentrations. The characteristic fall in blood glucose, reaching a nadir within hours of birth, was associated with a significant increase in glucagon concentration. Despite persistence of relative glucopenia, glucagon did not change appreciably between 2 and 24 h of life. A further significant elevation in glucagon concentration occurred from day 1 to day 3 of life associated with a return of glucose to euglycemic levels. In contrast to the sluggishness of pancreatic glucagon release, glucagon-like immunoreactivity rose markedly to mean levels of approximately 2,000 pg/ml after introduction of formula feeding. No significant changes in insulin levels were observed in these studies. Arginine infusion via an umbilical vein catheter into six infants within 6 h of birth elicited a brisk, almost threefold increment in glucagon concentration (from 339+/-85 to 940+/-254 pg/ml) in blood obtained from, or close to, the portal circulation. Bolus injection of alanine (1 mmol/kg) into a peripheral vein to six infants resulted in significant increments in glucagon (mean maximal, 128 pg/ml) as well as glucose and insulin. The observations suggest that spontaneous glucagon secretion may be an important factor in neonatal glucose homeostasis. Secretion seems more brisk in response to amino acid stimulation than to a falling glucose concentration.

Alanine↗
PubMedDate not supplied

Mechanisms of surfactant dysfunction in early acute lung injury.

Surfactant dysfunction that occurs during acute lung injury is associated with alterations in phospholipid, total protein, and surfactant apoprotein content. The functional importance of these changes was examined by characterizing the biophysical properties and biochemical composition of lung surfactant from endotoxin-treated guinea pigs (LPS) with acute lung injury. Static and dynamic lung compliance significantly decreased following endotoxin exposure. Lavage fluid demonstrated a neutrophil predominance, and tissue histopathology revealed inflammation consistent with acute lung injury. LPS surfactant isolated by ultracentrifugation had minimum surface tensions of 21 dynes/cm compared to 2 dynes/cm among control samples. Biochemical abnormalities in LPS surfactant included increased total protein, decreased phosphatidylcholine, and increased sphingomyelin, phosphatidylethanolamine, and lysophosphatidylcholine. The addition to normal guinea pig surfactant of butanol extracts precipitated from lavage fluid of LPS animals and containing known amounts of protein caused elevations in minimum surface tensions to > or = 20 dynes/cm at protein to phospholipid ratios equivalent to those observed in LPS surfactant pellets. Addition of equal amounts of precipitate isolated from control animals had no effect on interfacial properties. Furthermore, addition of lysophosphatidylcholine and sphingomyelin to normal surfactant to simulate composition changes observed in LPS surfactant had minimal effect on surface film behavior. The results support the hypothesis that aqueous soluble inhibitors of surfactant are generated within the alveolar compartment during acute inflammation, and that surfactant dysfunction cannot be accounted for on the basis of phospholipid composition changes.

Animals↗