The development of large-scale animal cell perfusion culture systems.
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Animals↗
Biomedical subjects
J Feder
Publications and source records attributed to J Feder.
Purification and characterization of a tissue plasminogen activator-inhibitor complex from human umbilical vein endothelial cell conditioned medium.
Tissue plasminogen activator-inhibitor complexes were purified from the conditioned medium of human umbilical vein endothelial cells by affinity chromatography followed by gel filtration. It was found that a single complex was isolated which can exist in two distinct interconvertible conformations. These may be separated by electrophoresis into a form with a 105,000 apparent molecular weight and a form with an 88,000 apparent molecular weight. The particular conformation which predominates may be altered by changing the pH at which preparations are incubated or by including dithiothreitol in incubation buffers. Plasminogen activator enzymatic activity may be partially recovered from purified complexes by incubation in the presence of fibrin. Incubation in 1.5 M NH4OH results in the dissociation of the complex into two major polypeptides of 67 and 40 kilodaltons (kDa). The 40-kDa protein was isolated by gel filtration high-pressure liquid chromatography. N-Terminal amino acid analysis of this protein revealed three distinct sequences. Two of these were nearly identical and matched the N-terminal sequence recently reported for the native plasminogen activator inhibitor from endothelial cells. The third sequence exactly matched an internal portion of the same protein. The results suggest that the internal sequence is located at the site where the inhibitor is cleaved by tissue plasminogen activator.
Amino Acid Sequence↗
How did Medicare's prospective payment system affect hospitals?
Using data from 1982 and 1984, we examined how Medicare's prospective payment system affected hospitals. The study showed that hospitals paid through the prospective payment system had significantly lower increases in Medicare costs and greater declines in Medicare use than did other hospitals. Unlike these other hospitals, for which Medicare costs approximately equaled Medicare revenues, hospitals receiving prospective payment kept Medicare costs from rising as fast as Medicare revenues, earning the profit that the prospective payment system allowed. The opportunity to earn a profit led hospitals to slow increases in Medicare costs, regardless of the level of revenue constraint. However, the more the prospective payment system constrained hospitals' revenues, the more hospitals slowed increases in Medicare costs. In the most constrained hospitals, slower increases in Medicare costs were accompanied by slower increases in total hospital spending. The least constrained hospitals slowed Medicare cost increases the least and did not show their overall spending. These hospitals nevertheless increased their profit margins the most, since the prospective payment system's federal rate paid them the highest rates relative to base-year costs. Since federal rates produced extra profits, not extra cost containment, their appropriateness is questionable. The prospective payment system should be modified to eliminate windfalls while continuing to promote cost containment.
Cost Control↗
Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells.
Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.
Animals↗
Flat bed hollow fiber reactors for large-scale cultivation of animal cells.
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Animals↗
Thermal properties of human IgG.
Dynamic light scattering experiments have been performed to study the aggregation kinetics of human immunoglobulin G (IgG). Aggregation and irreversible cluster growth results when IgG solutions (2-15 mg/ml) are heated above 50 degrees C. The measured scattering intensity I and effective hydrodynamic radius (R) can be described consistently by a Smoluchowski aggregation process. The number of clusters ni(t) containing i monomers at time t are computed. The radius of an i cluster is assumed to be Ri = R0 i beta, where beta is the cluster exponent. This kinetic process results in the following characteristic power law behavior: (R)/R0 = (1 + gamma R (T, C, c)t) alpha R and (I)/I0 = (1 + gamma 1 (T, C, c)t) alpha I. Here R0 = 5.51 nm, is the monomer hydrodynamic radius, and I0 the scattered intensity from the monomer solution at temperature T and concn C. A fraction, c approximately 0.48 of the IgG monomers are heat stable up to 63 degrees C and do not participate in the aggregation process. The power-law behavior of mean value of R/R0 and mean value of I/I0 indicates scaling, and indeed a very satisfactory data collapse results from our data. The best non-linear fit of the power-law forms gives alpha R = 0.48 +/- 0.05, alpha I = 1.00 +/- 0.01 and beta = 0.39 +/- 0.04. We also find that the heat aggregation of IgG is an activated process. Fits of the experimental data Gibbs free energy for the activated complex delta G* = 13.8 +/- 0.1 kcal/mole at 56 degrees C. The temp dependence of the growth rates exhibits an Arrhenius behavior with an enthalpy of activation delta H* = 120 +/- 5 kcal/mole.
Chromatography, Gel↗
The development of perfusion culture systems for anchorage-dependent animal cells.
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Animals↗
A threat or a promise: acquisition of teaching hospitals by investor-owned chains.
In the early 1980s, acquisition of a small number of teaching hospitals by investor-owned chains raised the spectre of a for-profit takeover of teaching institutions. Drawing on experience to date, as well as interviews with affected parties, this article assesses the likely scope of such acquisitions and their impact on the education, research, and indigent care that teaching hospitals provide. Our assessment concludes that relatively few teaching hospitals are likely to satisfy the financial criteria chains apply to acquisitions; that hospitals with modest rather than extensive commitments to education and research are most likely to satisfy these criteria; and that terms of sale typically enhance, rather than undermine, these institutions' resources for research, education, and, to a lesser extent, indigent care, at least in the short run. In the long run, continuation of these activities is more likely to be a function of third-party payment policies than of proprietary versus nonprofit hospital ownership.
Education, Medical↗
Medicare reform: nibbling at catastrophic costs.
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Catastrophic Illness↗
Cleavage site specificity of vertebrate collagenases.
A series of synthetic peptide substrates for vertebrate collagenase having the structure Ac-Pro-Leu-Gly-X-Leu-Gly-OC2H5, where X is Leu, Ile, Val, Phe and Ala, have been prepared. Collagenolytic enzymes from various sources cleave these substrates with differing relative rate patterns. This series of peptides should be valuable for characterization of collagenases.
Amino Acid Sequence↗
Determination of the number of endothelial cells in culture using an acid phosphatase assay.
A simple assay is described in which small numbers of endothelial cells in culture can be determined by measuring acid phosphatase activity. After removal of the growth medium from cells grown in 96-well culture plates, the cells are lysed in buffer containing the detergent Triton X-100 and the phosphatase substrate p-nitrophenyl phosphate. After 2 h at 37 degrees C, the reaction is stopped with sodium hydroxide, and color development is determined using a rapid multiwell plate reader. The assay detects 100 to 10,000 cells per well. The assay has been used to determine growth curves for endothelial cells in the presence and absence of endothelial cell growth factor from bovine hypothalamus and to monitor fractions during purification of the growth factor. Minor modifications in the assay allow it to be fully automated.
Acid Phosphatase↗
Chromosomal assignment of seven human genomic DNA sequences associated with restriction fragment length polymorphisms.
Seven phage clones containing human sequences were picked at random from a human genomic library cloned in Charon 4A. The clones are devoid of repetitive sequences and can be used to recognize restriction fragment length polymorphisms (Feder et al. 1985). The chromosomal locations of the sequences defined by the seven clones have been determined by Southern blotting and DNA hybridization to DNA from human-mouse somatic cell hybrids. The chromosomal assignment of these sequences should increase their value as genetic markers in family studies.
Animals↗
Transient inhibition of DNA synthesis results in increased dihydrofolate reductase synthesis and subsequent increased DNA content per cell.
We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.
Animals↗
Chronic mental patients in nursing homes: reexamining data from the National Nursing Home Survey.
A reanalysis of data from the 1977 National Nursing Home Survey, including data not available earlier, led to an estimate that 668,000 chronic mentally ill patients reside in nursing homes. Several subpopulations of nursing home residents were also identified and compared, which showed, for instance, that residents with only mental disorders were younger, were less likely to need the help of another person in daily activities, and were much less likely to be totally dependent than residents with only physical disorders. However, not surprisingly, mentally ill residents were more likely to have behavior problems and to have much longer stays. Residents diagnosed as senile, with or without a physical disorder, more closely resembled the purely physically ill than the purely mentally ill patients. The data illustrate the wide range of needs of mentally ill nursing home residents and reinforce the importance of assessing and improving the appropriateness of care offered.
Adaptation, Psychological↗
Effect of 1% sodium hyaluronate (Healon) on a nonregenerating (feline) corneal endothelium.
A series of experiments were performed to investigate the effect of 1% sodium hyaluronate (Healon) on the nonregenerating corneal endothelium of the cat. Aqueous humor replacement with 1% sodium hyaluronate resulted in mild, transient elevations of intraocular pressure compared to eyes that were injected with balanced salt solution. Sodium hyaluronate 1% protected the feline endothelium against cell loss incurred by contact with hyaluronate-coated intraocular lenses compared to endothelial contact with lenses that were not coated with sodium hyaluronate. The use of intraoperative 1% sodium hyaluronate, however, did not protect against endothelial cell loss incurred by penetrating keratoplasty or prevent subsequent skin graft-induced corneal homograft rejections. Homograft rejections were milder, however, in some eyes that received grafts coated with 1% sodium hyaluronate. Image analysis of photographs of trypan blue- and alizarin red-stained corneal buttons after trephining, stretching of Descemet's membrane, rubbing against iris-lens preparations, or immediately after penetrating keratoplasty demonstrated that the stretching of the posterior cornea is an important cause of endothelial damage that would not be protected against by a viscoelastic coating.
Animals↗
A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines.
We have previously reported that rodent tumor cell lines secrete a potent vascular permeability factor with a molecular weight of 34,000-42,000 (Senger et al. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science (Wash. DC), 219: 983-985, 1983). This tumor-secreted vascular permeability factor (VPF) causes a rapid and completely reversible increase in microvascular permeability in the species (guinea pig or rat) from which the tumors were derived without causing mast cell degranulation or endothelial cell damage or exciting an inflammatory cell infiltrate. This VPF may be responsible, at least in part, for the increased permeability which is commonly displayed by solid and ascites tumor vessels. We have now examined 7 human tumor cell lines and have determined that 5 of them also secrete this same VPF. Antibody raised to guinea pig line 10 VPF neutralized more than 90% of the vascular permeability-increasing activity secreted by these 5 human tumor lines. Furthermore, VPFs from both guinea pig and human tumor sources bound to and were eluted similarly from immobilized heparin and comigrated identically on sodium dodecyl sulfate-polyacrylamide gels. Finally, 2 tumorigenic (in nude mice) human cell lines were found to secrete at least 14-fold more VPF than their directly matched, nontumorigenic counterparts, suggesting that elevated expression of this permeability factor may correlate with neoplastic transformation. These data suggest that a broad spectrum of tumor cells from several species, including humans, secretes a highly conserved molecule that enhances local vascular permeability and that this function may be important for tumor growth.
Animals↗
Synthetic substrates of vertebrate collagenase.
The active site specificity of vertebrate collagenase was mapped with the synthesis of a variety of peptides, peptolides, and peptide esters. The enzyme was found to prefer very lipophilic sequences, and it was also found to be an esterase. The thio peptolide Ac-Pro-Leu-Gly-SCH[CH2CH(CH3)2]CO-Leu-Gly-OC2H5 was found to be an exceptional substrate. High-performance liquid chromatography and tandem mass spectrometry were used to unambiguously establish the cleavage site in several peptide substrates.
Animals↗
Fibrin-enhanced endothelial cell organization.
The formation of cloned bovine endothelial cells into capillary-like tubes is accelerated from 3-7 days to 2-18 h in the presence of fibrin. Indirect immunofluorescence showed the presence of both fibrin and fibronectin in the strands along which the cells organized. Electronmicroscopy revealed the same type of cell structures as form in the absence of fibrin; it also revealed a gradual decrease with time of the fibrin within the putative lumen. Fibrin and fibronectin are commonly present during angiogenesis in vivo, thus these in vitro observations may well have relevance to the in vivo process.
Agar↗