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Biomedical subjects

L K Cohen

Publications and source records attributed to L K Cohen.

At least 37 records · Page 2Linked to original sources
PubMed1993

Host range selection of vaccinia recombinants containing insertions of foreign genes into non-coding sequences.

A simple yet powerful selection system was developed for the insertion of foreign genes in vaccinia virus. The selection system utilizes the vaccinia virus K1L (29K) host range gene which is located in HindIII M. This gene is necessary for growth in RK-13 cells but not in BSC40 or CV-1 cells. A vaccinia mutant (vAbT33) unable to grow on RK-13 cells was constructed having sequences at the 3' end of the K1L gene and the adjacent M2L gene deleted and replaced with the beta-galactosidase gene regulated by the BamHI F (F7L) promoter. A recombination plasmid containing the hepatitis B surface (HBs) antigen gene regulated by the M2L promoter and the complete sequence of the K1L gene was used to insert the HBs gene into vAbT33. The M2L negative K1L positive recombinant was easily isolated in two rounds of plaque purification by plating the virus on RK-13 cell monolayers. The K1L gene selection system allows the isolation of recombinants arising at frequencies as low as 1/100,000. It was noted that recombinants containing vaccinia sequence duplications (promoters) resulted in intragenomic recombinations that eliminated all sequences between the duplications. A second recombination plasmid was constructed that allowed insertion into the vaccinia genome without the loss of vaccinia coding sequences. This was achieved by insertion of the pseudorabies virus GIII gene regulated by the vaccinia H5R (40K) promoter between the translation and transcription stop signals at the 3' end of the K1L gene. The K1L gene transcription stop signal thus became the stop signal for the inserted GIII gene and an upstream transcription stop signal present in the H5R promoter fragment provided the stop signal for the K1L gene. This manipulation of the vaccinia genome had no effect on the accumulation or 5' end of the M2L gene transcripts. Although the insertion lengthened the 3' end and lowered the accumulation of K1L transcripts it altered neither the virulence nor the immunogenicity of the recombinant.

Base Sequence↗
PubMed1992

Molecular attenuation of vaccinia virus: mutant generation and animal characterization.

These studies demonstrated that the inbred BALB/c mouse strain can be optimized for the assessment of vaccinia virus virulence, growth, and spread from the site of inoculation and immune protection from a lethal vaccinia virus challenge. The studies established that manipulation of the vaccinia virus genome generated mutants exhibiting a wide range of attenuated phenotypes. The nine NYCBH vaccinia virus mutants had intracranial 50% lethal doses that ranged from 2 to greater than 7 log10 units. The decreased neurovirulence was due to decreased replication in brain tissue. Three mutants had a decreased ability to disseminate to the lungs, brains, livers, and spleens of mice after intranasal infection. One mutant had a decreased transmission from mice infected by tail scarification to naive cage mates. Although the mutants, with one exception, grew to wild-type titers in cell culture, they showed a growth potential on the scarified skin of mice that was dramatically different from that of the wild-type virus. Consequently, all of the mutants had significantly compromised immunogenicities at low virus immunization doses compared with that of the wild-type virus. Conversely, at high immunization doses most mutants could induce an immune response similar to that of the wild-type virus. Three Wyeth vaccine strain mutants were also studied. Whereas the thymidine kinase, ribonucleotide reductase, and hemagglutinin mutants had a reduced virulence (50% lethal dose), only the thymidine kinase mutant retained its immunogenicity.

Administration, Intranasal↗
PubMed1991

Formation of lentivirus particles by mammalian cells infected with recombinant fowlpox virus.

Recombinant fowlpox viruses (FPV) containing the env or gag-pol genes of simian immunodeficiency virus from macaques (SIVmac) were constructed. The env, gag, and pol-encoded polypeptides were efficiently expressed and processed in avian cells productively infected with FPV as well as in mammalian cells, in which FPV infection is abortive. In addition, the recombinant FPV expressing the gag-pol genes directed the formation of defective, lentivirus-like particles which were released into the culture medium of infected cells. Coinfection of cells with the env and gag-pol recombinant viruses resulted in the generation of particles containing SIVmac envelope glycoprotein. The applications of this system to vaccine development are discussed.

Animals↗
PubMed1991

Utilization of DNA recombination for the two-step replacement of growth factor sequences in the vaccinia virus genome.

An efficient procedure for the generation of sequence-specific alterations of the vaccinia virus genome was demonstrated. Homologous DNA recombination within cells infected with vaccinia virus was used for the deletion or replacement of promoter sequences of the viral growth factor gene by a procedure comparable to transplacement in Saccharomyces cerevisiae. This DNA replacement procedure can potentially be used to generate any sequence alteration within the vaccinia virus genome. Deletion of growth factor promoter sequences resulted in a dramatic reduction in growth factor gene transcription and protein synthesis. Replacement of growth factor promoter sequences with promoter sequences of the strong constitutive 40-kDa gene resulted in an increase in gene transcription and protein synthesis and an altered temporal pattern of expression. Virus containing mutations in the growth factor gene demonstrated different plaque morphologies on cell culture monolayers.

Base Sequence↗
PubMed1990

Promoting oral health: guidelines for dental associations.

Dental associations throughout the world face many new challenges. For example, many industrial countries confront the potential of excess manpower to meet current demands, while developing countries try to cope with scarce resources to control existing and increasing disease levels. This guide is intended to facilitate the development of oral promotion programmes in both situations. In industrialized countries, an oral health promotion programme should increase awareness and interest among consumers, thus facilitating the conversion of unmet need to demand. In developing countries, the oral health promotion guidelines demonstrate ways to extend resources to meet a broader base of need. In all countries, oral health promotion can demonstrate the benefit of self-care and emphasize the consumer's responsibility for personal health. Dental associations enjoy a long history of promoting oral health, yet developing a coherent programme or realizing the results of these efforts can sometimes be disappointing or frustrating. The guide is organized around a recommended sequence of activities: policy formation and dissemination; planning group; information gathering; goal setting; strategic planning; implementation; and evaluation. This document presents guidelines, not recipes. Given the range of economic, social and health needs of the Fédération Dentaire Internationale member countries, a recipe would be inappropriate. These guidelines can be tailored to the needs of your association based on the oral health needs of your population. They can be used to determine new or modified policies, to assess long- and short-term goals, to monitor progress and to educate the membership. Case studies derived from actual experiences of dental associations as they planned and analysed their efforts to promote oral health are presented. This information is included to highlight different approaches as well as problems encountered. The case studies illustrate various parts of the planning implementation and evaluation process, while the text should serve as a comprehensive review of all these components.

Australia↗
PubMed1990

Leadership role of dental associations: oral health promotion.

'Promoting Oral Health: Guidelines for Dental Associations' is the product of Working Group 3 on Oral Health Promotion of the Commission on Oral Health, Research and Epidemiology of the FDI. This paper describes the guidelines document, its rationale and its potential utility. An organized planned sequence of activities, including policy formation and dissemination, planning group structure and function, information gathering, goal setting, strategic planning of objectives and interventions, implementation as well as monitoring and evaluation, are reviewed. Relevant to both industrialized and developing countries, these oral health promotion guidelines can be used to develop programmes to demonstrate the benefit of self-care and appropriate demand for dental services.

Health Planning↗
PubMed1989

Advances in the epidemiological study of oral-facial diseases.

Both demographic patterns and disease distribution are changing rapidly in the United States. These developments have led to the recognition that the epidemiology of many conditions is poorly understood, and that other research has thus been hindered. Four areas of epidemiological study were chosen for detailed analysis of how new technology will affect the conduct of future research. These areas, selected because information about them will be increasingly needed in an aging society, were periodontitis, temporomandibular disorders (TMD) and other orofacial pain, salivary gland disturbances, and health services research. The potential effect of new technology was examined in the short, intermediate, and long term. While the nature of epidemiological study is unlikely to change with the advent of new technology, the scope of potential studies will become broader. Advances in diagnostic techniques from elsewhere will permit far more precise diagnosis than is possible at present. Computer technology will permit an efficient system of epidemiological surveillance to provide current data on trends in tooth loss, caries, and periodontitis--data which will complement the results of national surveys. Analytical studies to produce hypotheses on the etiology of oral conditions, especially in such poorly-understood areas as chronic pain and TMD, will help direct clinical research in those areas.

Computers↗
PubMed1988

Delineation of the viral products of recombination in vaccinia virus-infected cells.

Plasmids containing the vaccinia virus thymidine kinase gene, its flanking DNA sequences, and the Escherichia coli beta-galactosidase gene were used in conjunction with a thymidine kinase-deficient virus to examine the viral products of recombination. Progeny derived from single-crossover events could be distinguished from those generated by gene conversion or double-crossover events when the beta-galactosidase gene was separated from the thymidine kinase gene by the flanking sequences. Using methotrexate to select for recombinant virus and a chromogenic indicator to detect beta-galactosidase, the generation of viral recombinants was measured over a 48-h period. Recombinant progeny were first observed at 12 h and increased to a maximum of 2.5% at 48 h. Single-crossover products, as determined by beta-galactosidase expression, reached a maximum of 57% of the recombinant population at 24 h and thereafter declined. DNA hybridization analysis was used to examine genomic structures of the progeny of the initial viral plaques, plaques purified three times, and those subject to a 10(4)-fold amplification. These analyses confirmed that single-crossover events within either the 5'- or 3'-homologous flanking sequences generated unstable recombinant structures. These structures were shown to contain a single copy of the intact thymidine kinase gene within the corresponding copy of the duplicated thymidine kinase flanking sequences, separated by the beta-galactosidase gene and plasmid DNA. Significantly, these duplicated structures could undergo further recombination to produce repeats of either the intact or the deleted thymidine kinase sequences. These intermediate structures ultimately degenerated to produce either the parental thymidine kinase-deleted or the wild-type genome. The wild-type genome was also shown to be generated directly by gene conversion or double-crossover events.

Cell Line↗
PubMed1987

Converting unmet need for care to effective demand.

Strategies to lower the barriers to both the receipt of personal dental services and to self-care are discussed in the light of the supporting body of socio-dental research. Planning to reach populations in need of care but who have not obtained such care involves both external marketing strategies as well as subsequent internal marketing strategies. Modifications of elements in the dental care delivery system, and approaches which impact on personal lifestyles of populations in need of care as well as on their environments, constitute a comprehensive marketing plan.

Delivery of Health Care↗
PubMed1984

Identification and mapping of polypeptides encoded by the P3HR-1 strain of Epstein-Barr virus.

The Epstein-Barr virus (EBV)-specified polypeptides induced upon viral replication in the P3HR-1 cell line have been examined by immunoprecipitation with a high-titer human anti-EBV serum. Twenty-five predominant polypeptides were identified in cell extracts, whereas 18 polypeptides were precipitated from cell-free translation reactions directed by total mRNA. Hybrid selection of mRNA to the BamHI DNA clones of the EBV genome and immunoprecipitation of the corresponding cell-free translation products revealed 98 EBV-specified polypeptides and their coding location along the viral genome. In addition, the viral polypeptides that bind reversibly to DNA-cellulose have been characterized and the deduced map locations of this functional group of EBV-specified polypeptides is presented.

Cell Line↗
PubMed1984

Identification of the DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus.

The DNA sequences encoding the large subunit of the mRNA-capping enzyme of vaccinia virus were located on the viral genome. The formation of an enzyme-guanylate covalent intermediate labeled with [alpha-32P]GTP allowed the identification of the large subunit of the capping enzyme and was used to monitor the appearance of the enzyme during the infectious cycle. This assay confirmed that after vaccinia infection, a novel 84,000-molecular-weight polypeptide corresponding to the large subunit was rapidly synthesized before viral DNA replication. Hybrid-selected cell-free translation of early viral mRNA established that vaccinia virus encoded a polypeptide identical in molecular weight with the 32P-labeled 84,000-molecular-weight polypeptide found in vaccinia virions. Like the authentic capping enzyme, this virus-encoded cell-free translation product bound specifically to DNA-cellulose. A comparison of the partial proteolytic digestion fragments generated by V8 protease, chymotrypsin, and trypsin demonstrated that the 32P-labeled large subunit and the [35S]methionine-labeled cell-free translation product were identical. The mRNA encoding the large subunit of the capping enzyme was located 3.1 kilobase pairs to the left of the HindIII D restriction fragment of the vaccinia genome. Furthermore, the mRNA was determined to be 3.0 kilobases in size, and its 5' and 3' termini were precisely located by S1 nuclease analysis.

Animals↗
PubMed1983

An unusually high-titer human anti-Epstein Barr virus (EBV) serum and its use in the study of EBV-specific proteins synthesized in vitro and in vivo.

Sera from a patient with a chronic Epstein Barr virus (EBV) infection contained unusually high anti-EBV antibody titers (1:2560 to 1:10,240 for EA(D) and 1:5,120 to 1:40,960 for VCA). One of these serum samples was shown by immunoprecipitation to recognize at least 11 EBV-specific proteins from virus producer cells labeled in vivo and 10 EBV-specific proteins from in vitro translations of producer cell mRNA. Six of the in vivo labeled proteins (135,000, 89,000, 50,000 to 55,000 doublet, 46,000, and 34,000 daltons) are "early" by their resistance to phosphonoacetic acid, and five (350,000, 220,000, 160,000, 140,000, and 85,000 daltons) are "late" membrane and capsid proteins. The EBV-specific proteins immunoprecipitated from in vitro translations had molecular masses of 150,000, 140,000, 115,000, 52,000, 50,000, 45,000, 34,000, 29,000, 17,000, and 15,000. Subcellular fractionation studies of cells labeled in vivo revealed that the 135,000-dalton protein and part of the 50,000 to 55,000 dalton protein doublet were found in both the nuclear and the cytoplasmic fractions, and thus are good candidates to be components of the EA(D) diffuse-type immunofluorescence observed with many EA-positive sera.

Adult↗
PubMed1981

The non-detergent solubilization and isolation of intracytoplasmic membrane polypeptides from Rhodopseudomonas sphaeroides.

A nonspecific solubilization of the intracytoplasmic membrane proteins from Rhodopseudomonas sphaeroides has been achieved in the absence of detergents. Following removal of the photopigments and phospholipids by acetone/methanol extractions, the protein is quantitatively solubilized in 6 M guanidine thiocyanate which is subsequently exchanged by dialysis for 7 M urea. A urea-soluble protein fraction is obtained which represents 80% of the initial protein and qualitatively contains all the polypeptides of the membrane resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the exception of the reaction center polypeptides M and L, and the 35.2-kilodalton species. Fractionation of the urea-soluble polypeptides on a Sepharose CL-6B column resolves into separate fractions the two major low molecular weight polypeptides identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Further examination of these fractions by isoelectric focusing and electrophoresis at pH 4.5 in the presence of 8 M urea reveals the presence of three major species each of which has been purified to homogeneity by a combination of ion exchange chromatography and preparative electrophoretic techniques. It is believed that these three major polypeptides are components of the light-harvesting pigment-protein complexes.

Cell Membrane↗
PubMed1981

Characterization of the three major intracytoplasmic membrane polypeptides isolated from Rhodopseudomonas sphaeroides.

The characterization of the three major membrane polypeptides indicated to be subunits of the light-harvesting bacteriochlorophyll-protein complexes of Rhodopseudomonas sphaeroides is described. Molecular weight determinations of the three polypeptides have been performed by sedimentation analysis and electrophoretic mobility and the values obtained were in agreement with each other as well as with those derived from compositional studies. NH2- and COOH-terminal amino acid determinations were performed for each of the species; two of the polypeptides produces no reaction with dansyl chloride and were concluded to have blocked amino termini. In each instance, the data were consistent with each of the polypeptides being a single, pure species. Further characterization of the proteins was accomplished by amino acid compositional analysis and peptide mapping of tryptic and chymotryptic digestions of the polypeptides. The number of peptides obtained from the digestions was in agreement with the amino acid compositional data. Two of the polypeptides, 15B and 15C, which possess very similar molecular weights, identical carboxyl termini and apparently blocked amino termini were shown by amino acid analysis and peptide mapping to be structurally discrete species, although related. The relationship between these purified proteins and the known activities and characteristics of the light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides is discussed.

Amino Acids↗