Biomedical subjects
D Korn
Publications and source records attributed to D Korn.
Reengineering academic medical centers: reengineering academic values?
Academic medicine is entering an era of profound, unsettling change resulting not simply from the drastic transformation of the health care marketplace but more fundamentally from the chronic, growing gap between academic medicine's seemingly insatiable demand for total resources and the supply of resources that society is willing to provide. To examine this problem, the author reviews the major factors that have shaped the development of academic medical centers (AMCs) since World War II and are now the roots of their vulnerability. The first was the major federal investment in university-based programs of science research and education that began in the 1940s; the second was the enactment in the 1960s of the Medicare/Medicaid legislation that established federal responsibility for the support of graduate medical education. After describing important characteristics (e.g., number of faculty, number of students, dollars spent on research) of the growth and accomplishment that resulted from this massive infusion of federal funds over the last few decades, the author discusses several adverse consequences, such as the de-emphasis on education in favor of research and clinical service delivery and the serious disjunction between the internal labor markets of the AMCs and the external labor markets of the real world that AMCs' graduates enter. The author then analyzes the severe challenges being faced by academic medicine in research, education, and clinical practice in the emerging resource-limited environment. Of particular concern are the fate of the clinical investigator and the future of clinical research. The author concludes with a list of four feasible strategic options for AMCs (e.g., "build one's own system") and an extensive list of what he believes AMCs will do to respond to the stresses now upon them (e.g., capitalize on unique strengths rather than trying to compete in all areas). He concludes that it will take courage for AMCs to preserve their core values in the new era, but that this can be done if AMCs craft new adaptive structures that are better attuned to the new environment and not wedded to one that is vanishing.
Preserving medical schools' academic mission in a competitive marketplace.
To gain a better understanding of the effects on medical schools of transformations in medical practice, science, and public expectations, the AAMC in 1994 formed the Advisory Panel on the Mission and Organization of Medical Schools and appointed six working groups to address relevant issues. This article is a report of the findings of the Working Group on Preserving Medical Schools' Academic Mission in a Competitive Marketplace, which was charged with exploring how medical schools could acquire and/or preserve an adequate patient base for teaching, research, and income generation in a competitive marketplace. The other groups' reports will appear in future issues of Academic Medicine. To understand the diversity of approaches that schools have taken to achieve this goal and to preserve their missions, the group interviewed representatives of nine medical schools, selected to represent a cross section of U.S. medical schools. The interviews took place on four occasions between June 1995 and March 1996. The information and comments shared by participants helped the working group gain insight into the fundamental issues it had been charged to address, including those of new delivery structures, what value schools offer to delivery structures, how education and research can be incorporated and supported financially, possible new pressures on relationships between medical schools and teaching hospitals, changes in faculty physicians' employment relationships and terms, and the role of the medical school in graduate medical education. In collecting and analyzing the data, the working group focused on the distinction between protecting an institution's existing enterprise and preserving an institution's core mission. This article gives a detailed overview of the information and comments each school presented, organized under the appropriate question. The working group's conclusions and commentaries on the findings follow. An appendix presents more detailed summaries of the schools' presentations, organised as case studies. The picture that emerges is complex. The working group concluded that medical schools will take a variety of approaches to define and preserve their missions. Most, but not all, medical schools will be able to secure the patient bases necessary to fulfill their missions even in a competitive marketplace. However, the nature of many of the schools is likely to change, and it is not clear whether the core missions of education and research will continue at their present levels at all schools.
Effects of various hygiene procedures on the surface characteristics of titanium abutments.
The use of cleaning instruments on titanium implants may cause undesired surface alterations. In a qualitative and quantitative assessment of these alterations, 5 titanium implant abutments were treated with a steel curet, a prototype pure titanium curet, an air abrasive polishing system, and an ultrasonic system. Custom-made polymer templates, used to secure the curet to a vertical guide bar and a spring scale to maintain a constant instrument pressure, guaranteed a standardized procedure and reproducible results. The ultrasonic and the air abrasive polishing method were also standardized. Evaluation by scanning electron microscopy (SEM) revealed surface alterations for all instruments and systems except the plastic curet, which did not roughen the surface at all. The confocal laser-scanning microscope allows a 3-dimensional reproduction of these surface alterations and their direct measurement. The profilometric tracing was not sensitive enough to register the minor effects caused by the titanium curet and the air abrasive polishing system. Dimensions of the resulting surface microstructure could be determined with the laser-scanning microscope. Since the influence of such surface defects on the peri-implant tissue reaction is unpredictable, the titanium curet and the air abrasive system can only be recommended with restrictions. The steel curet and the ultrasonic system proved to be totally unsuitable for cleaning titanium implants.
The JIM interview. David Korn, MD.
When David Korn, MD, was named dean of the Stanford University School of Medicine on October 9, 1984, he assumed leadership of a world class research institution. Stanford was at the forefront of medicine in the areas of transplantation and oncology, and the steady influx of privately insured patients had generated a net operating surplus of $17 million in that year alone. However, in the same issue of the Stanford University Hospital newsletter which announced the selection of Korn as Dean, a small article appeared on a new prospective payment system based on diagnosis-related groups (DRGs). The article stated that the new system had begun smoothly, though some payments for cost outliers had been delayed. Other cost containment measures soon followed, most notably the implementation of managed care, and by 1990, Stanford was $14 million in the red. Buffeted by changes in medical reimbursement, competition with less costly hospitals, and a nasty squabble with Congress over indirect research costs, Stanford has been on the frontlines of a struggle now confronting many academic medical centers. After successfully consolidating the university's clinical services into a unified Stanford Health System, Korn announced that he would be stepping down as Dean on April 1. Interviewed in his office in Palo Alto, Korn reflected on the difficulties of dealing with managed care, the current financial state of the institution, and what Stanford's experience may predict for other academic medical centers.
Funding for cancer centers: a challenge of scarce resource allocation.
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Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases.
The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.
The evolutionary conservation of DNA polymerase alpha.
The evolutionary conservation of DNA polymerase alpha was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase alpha monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. The most highly cross-reactive antibody recognizes a unique epitope on a 165-180 kDa catalytic polypeptide in cell lysates from several eukaryotic sources, as distant from man as the amphibians. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase alpha catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA polymerase alpha among vertebrates and the conservation of specific genetic sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.
Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases.
We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide. Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation. Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus. Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction. Two putative DNA interacting domains are also identified.
Gene expression of human DNA polymerase alpha during cell proliferation and the cell cycle.
We studied the expression of the human DNA polymerase alpha gene during cell proliferation, during cell progression through the cell cycle, and in transformed cells compared with normal cells. During the activation of quiescent cells (G0 phase) to proliferate (G1/S phases), the steady-state mRNA levels, rate of synthesis of nascent polymerase protein, and enzymatic activity in vitro exhibited a substantial and concordant increase prior to the peak of in vivo DNA synthesis. In transformed cells, the respective values were amplified greater than 10-fold. In actively growing cells separated into discrete stages of the cell cycle by counterflow elutriation or by mitotic shakeoff, levels of steady-state transcripts, translation rates, and enzymatic activities of polymerase alpha were constitutively and concordantly expressed at all stages of the cell cycle, with only a moderate elevation prior to the S phase and a slight decline in the G2 phase. These findings support the conclusion that the regulation of human DNA polymerase alpha gene expression is at the transcriptional level and strongly suggest that the regulatory mechanisms that are operative during the entrance of a cell into the mitotic cycle are fundamentally different from those that modulate polymerase alpha expression in continuously cycling cells.
Structural and enzymological characterization of immunoaffinity-purified DNA polymerase alpha.DNA primase complex from KB cells.
We describe the polypeptide structure and some of the catalytic properties of a DNA polymerase alpha.DNA primase complex that can be prepared from KB cells by immunoaffinity purification. The procedure is based on monoclonal antibodies that were raised against a biochemically purified, catalytically active core protomer of the polymerase. In all respects tested, the basic mechanism of substrate recognition and binding by the immunoaffinity-purified polymerase is qualitatively identical to that of the core protomer. The immunoaffinity-purified KB cell polymerase alpha X DNA primase is structurally complex. On the basis of extensive immunochemical analyses with five independent monoclonal antibodies, three of which are potent neutralizers of polymerase alpha activity, peptide mapping studies, and the application of a sensitive immunoassay that permits detection of polymerase alpha antigens in crude cell lysates, we have established that the principal form of catalytically active DNA polymerase alpha in KB cells is a phosphoprotein with a molecular mass of 180 kilodaltons. This protein is stable in vivo, with an estimated half-life of greater than or equal to 15 h. In contrast, the polypeptide is extremely fragile in vitro and generates partial degradation products of p165, p140, and p125 that explain the "microheterogeneity" typically exhibited by polymerase alpha peptides in denaturing polyacrylamide gels. In addition to the catalytically active polymerase alpha polypeptide(s), the immunopurified enzyme fraction typically contains three other proteins, p77, p55, and p49, the functions of which have not yet been established. These proteins do not display polymerase alpha epitopes and have been shown by peptide mapping to be independent species that are unrelated either to the large polymerase peptides or to one another. The polypeptide p77 is also a phosphoprotein, and in both p180 and p77 the phosphorylated amino acids are exclusively serine and threonine.
Monoclonal antibodies against human DNA polymerase-alpha inhibit DNA replication in permeabilized human cells.
Monoclonal neutralizing antibodies against DNA polymerase-alpha substantially inhibit nuclear DNA replication in lysolecithin-permeabilized cultured human fibroblasts. The degree of inhibition of DNA synthesis is proportional to antibody concentration, and the effect is specific in that RNA synthesis measured under the same experimental conditions is unperturbed. Autoradiographic data demonstrate that the magnitude of the inhibition measured in the mass culture reflects the uniform response of all the constituent cells in the target population. These observations confirm the participation of DNA polymerase-alpha in replicative DNA synthesis and identify a versatile, novel approach to the dissection of mammalian processes of DNA replication and repair.
Assignment of the gene for human DNA polymerase alpha to the X chromosome.
We have applied an assay based on a monoclonal antibody that discriminates the activity of human DNA polymerase alpha in rodent-human somatic cell hybrid clones to identify a single genetic locus that is both necessary and sufficient for the expression of DNA polymerase alpha. We have mapped this locus to the short arm of the human X chromosome, near the junction of bands Xp21.3 and Xp22.1, and demonstrated that it is not expressed from an inactive X chromosome.
DNA primase from KB cells. Evidence for a novel model of primase catalysis by a highly purified primase/polymerase-alpha complex.
The oligonucleotide primers synthesized by a highly purified KB fraction containing DNA primase and DNA polymerase-alpha activities display reproducible alterations of structure and composition in response to dNTPs. These observations are sufficiently explained by a novel model of primase catalysis that defines the primase as a complex enzyme with distinct catalytic properties that are regulated by mechanisms exquisitely sensitive to dNTP concentration. The enzyme performs the template-directed, de novo synthesis of 1 unit of oligoribonucleotide (canonical priming reaction) and then elongates that moiety to a limited extent by several cycles of addition of short tracts of homogeneous oligoribonucleotide or oligodeoxynucleotide. The oligomeric units have modal lengths of approximately 11- to 14- nucleotides that are postulated to reflect the inherent processivity of the catalytic mechanisms. Elongation is accomplished by two catalytic centers, or conformers of a single center, that are synchronously coupled, mutually exclusive, and extremely stringent for respective rNTP and dNTP substrates. Transitions between these two catalytic modes are regulated by dNTPs and demonstrable at dNTP:rNTP concentrations of less than or equal to 10(-4). In the absence of dNTPs, the primase produces a family of oligoribonucleotides, approximately 24- to 36- nucleotides long; at dNTP levels between 0.08 and 0.80 micron, the primase synthesizes mixed oligomers composed of strictly alternating tandem arrays of oligoribo- and oligodeoxynucleotide units; and at dNTP levels greater than or equal to 4.8 micron, the primase becomes stabilized in the deoxy mode after the initial oligoribo leads to oligodeoxy transition and products contain only a single 5' -terminal unit of oligoribonucleotide. The model predicts that the physiologically significant primer for DNA polymerase-alpha is a mixed 5' -oligoribo-3' -oligodeoxynucleotide and the signal which governs the switch from RNA leads to DNA synthesis is intrinsic in the primase mechanism and is generated by ambient dNTPs.
DNA primase from KB cells. Characterization of a primase activity tightly associated with immunoaffinity-purified DNA polymerase-alpha.
A very highly purified fraction of KB cell DNA polymerase-alpha, prepared with a monoclonal antibody, contains DNA primase activity. The primase synthesizes oligonucleotide chains initiated with ATP in a reaction that is resistant to alpha-amanitin and strictly dependent on added template and ribonucleoside triphosphates (rNTPs). In the presence of added dNTPs and M13 DNA template, the primase produces a uniform population of oligoribonucleotides, predominantly hexamers to decamers, that are extended by polymerase-alpha into DNA chains up to 3000 nucleotides long. There is no evidence for nucleotide preferences at RNA/DNA junctions. In the absence of added dNTPs, the oligomeric products are heterogeneous in size and composition and susceptible to cleavage by pancreatic DNase I due to their content of short oligodeoxynucleotide tracts synthesized by primase from trace contaminant dNTPs in the rNTP substrates. The primase and polymerase-alpha activities are distinguishable by several physical and chemical criteria, and the primase reaction is only partially sensitive to two potent, independent monoclonal antibodies that neutralize polymerase-alpha. Although the presence of both primase and polymerase-alpha activities in a highly purified immune complex prepared with a monoclonal antibody argues for their tight physical association, the chemical, physical, and immunological discriminations indicate the two catalytic entities are functionally and structurally distinct.
Monoclonal antibodies against human DNA polymerase-alpha do not cross-react with glucocorticoid receptors.
None of 16 independent monoclonal antibodies against human (KB cell) DNA polymerase-alpha recognizes epitopes on cytoplasmic glucocorticoid receptors prepared from the same cells. Consistently negative results are obtained with separate assays that measure antibody binding to uncharged receptors or to charged receptor complexes that have been preloaded with a specific steroid ligand. These results must qualify the interpretation of possible immunological relations between polymerase-alpha and glucocorticoid receptors that were inferred from studies with polyclonal antisera of poorly defined specificity.
Tight association of DNA primase with a subspecies of mouse DNA polymerase alpha.
Evidence was obtained for tight association of DNA primase activity with a subspecies of mouse DNA polymerase alpha by study with immunoadsorption assay using two monoclonal antibodies specific for human DNA polymerase alpha that have been shown to react with mouse murine myeloma DNA polymerase alpha (Tanaka, S., Hu, S.-Z., Wang, T.-S.-F. & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390). This result was supported by the finding that ethidium bromide at concentrations of less than 20 microM somewhat stimulated the syntheses of DNA and initiator RNA on unprimed poly(dT) by the novel subspecies of DNA polymerase alpha, but strongly inhibited DNA synthesis with poly(dT) X oligo(rA), suggesting that the conversion of synthesis from initiator RNA to DNA is continuous. Furthermore, the results of neutralization assay with the antibodies and experiment using aphidicolin suggested that the primase site is functionally distinguishable from the catalytic site of DNA polymerase activity.
Intracellular localization of human DNA polymerase alpha with monoclonal antibodies.
We have successfully established 16 stable murine hybridomas that secrete monoclonal antibodies specific for human DNA polymerase alpha. The results of immunocytochemical studies, using 4 of these monoclonal antibodies and immunoperoxidase detection methods, document the exclusively intranuclear localization of DNA polymerase alpha in three separate lines of cultured human cells. By light microscopy, the immunoperoxidase reaction product exhibits a diffuse pattern of distribution within the nucleoplasm, but nucleoli are clearly negative. In cultures of the transformed lines, KB nd BeWo, more than 955 of the cells are positive, suggesting that intranuclear DNA polymerase alpha antigens persist throughout the mitotic cycle. In striking contrast, in the normal diploid fibroblast line, WI-38, a smaller fraction of the cultured cells is positive, and there is no detectable polymerase alpha antigen in the closely apposed cells of microcolonies that are presumed to be contact-arrested and no longer mitotically cycling. In cells in mitosis that have dissolved their nuclear envelopes (and are thus transiently anucleate), the anti-polymerase alpha reaction continues to be strongly positive, and in this single circumstance the reaction product is diffusely distributed throughout the cell cytoplasm. Initial electron microscopic examination of KB cells confirms and extends these observations. The immunoperoxidase reaction product is essentially limited to the nuclear compartment and is predominantly distributed in the midzonal region of the nucleoplasm between centrally disposed nucleoli and peripherally located blocks of condensed chromatin.