Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.

DNA isolated from defective and nondefective virions of herpes simplex type 1 (HSV-1) (strain Patton) was digested with restriction endonucleases, and the resulting DNA fragments were inserted in the EK2 coliphage vector lambdagtWES . lambdaB. The recombinant DNA was encapsidated in vitro under P4 maximum containment conditions. These lambda-HSV1 hybrids were purified and amplified, and the DNA was isolated in the P4 facility. DNA, free of viable phage and bacteria, was removed from P4 conditions and analyzed. Represented among the hybrids studied to date are DNA fragments from about 50 percent of the normal HSV-1 genome. The hybrids derived from defective HSV-1 DNA fragments demonstrate the existence of many similar but not identical classes of defective genomes.

Expression of anionic glutathione-S-transferase and P-glycoprotein genes in human tissues and tumors.

The development of multidrug resistance in MCF-7 human breast cancer cells and the acquisition of broad resistance to xenobiotics in rat hyperplastic nodules are both associated with increased P-glycoprotein (mdr) gene expression as well as changes in activities of intracellular detoxication enzymes; among these changes is a significant increase in the activity of the anionic isozyme of glutathione-S-transferase (GST). We have isolated a cDNA encoding the human anionic glutathione-S-transferase, GST pi-1, from a cDNA library constructed from multidrug-resistant MCF-7 cells. The deduced amino acid sequence of GST pi-1 shows that while the human anionic GST displays 85% nucleotide and amino acid sequence homology to the rat anionic isozyme, it is markedly less related to human basic GST isozymes. We have examined the expression of GST pi and P-glycoprotein in 170 specimens of human tissues and tumors. P-Glycoprotein RNA expression was positive in eight of 23 lymphomas and two of 12 colon tumors; however, many other normal and malignant tissues, including lung, bladder, and breast tumors, had low or undetectable levels of P-glycoprotein RNA expression. In contrast, GST pi was readily detected in a wide variety of normal and malignant tissues. The level of GST pi mRNA expression in normal tissues was heterogeneous, with lowest levels found in liver and the highest levels found in lung, esophagus, and placenta. GST pi was also variably expressed in human tumors, with the lowest relative levels occurring in lymphoma and breast cancer and the highest levels found in lung cancer and head and neck tumors. In addition, comparison of paired specimens from the same patient indicated that GST pi expression was increased in many tumors relative to matched normal tissue.

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles.

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.

Restructuring the CNS role for a managed care environment.

The CNS role in a large northeast teaching hospital was the focus of a major organizational change process in response to a tightening fiscal environment. CNSs worked with the nurse executive team to restructure their role from expert consultant to case manager, nurse manager partner, and resource and consultant to the Department of Nursing. Development needs of the CNSs were assessed before and after role revision to evaluate the process and provide evidence of learning and change. The change and learning process evidenced in this article exemplifies the CNSs' ability to adapt and respond to the rapidly changing needs of today's managed care setting.

Strengthening of a model composite restoration using shape optimization: a numerical and experimental study.

OBJECTIVE: This study aims to validate a cavity shape optimization approach for improving the debonding resistance of dental restorations by carrying out fracture tests on restored model teeth with standard and optimized cavity designs. METHOD: The bio-mimetic stress-induced material transformation (SMT) optimization method was incorporated into the finite element (FE) program ABAQUS as a user material (UMAT) subroutine. The method uses stress minimization to optimize the cavity shape of a MOD restoration in an artificial premolar with special reference to the tooth-restoration interface under occlusal loads. The mechanical performance of the optimized design was first verified through FE analysis and then compared with that of the conventional design using fracture tests on model teeth. RESULTS: The SMT optimization process indicated a T-shape cavity as a more favorable design for the MOD restoration in the artificial premolar. Compared with the conventional parallel wall, or undercut design, the T-shape cavity was shown numerically to reduce the interfacial stresses by up to 69%, and experimentally to increase the mean debonding resistance of the model teeth by 23% (p<0.05). SIGNIFICANCE: Cavity shape optimization can help increase the debonding resistance of restored teeth by reducing the interfacial stresses between tooth and restoration under occlusal loads.

"Running the vicious cycle backward" and other systems solutions to nursing problems.

Peter Senge's book, The Fifth Discipline: The Art and Practice of the Learning Organization, is a business bestseller. The fifth discipline refers to systems thinking, a conceptual framework for making the full pattern of complex, interrelated actions clearer and helping people see how to change them. This article summarizes selected key concepts from Senge's work and explains how nurse executives can use systems thinking to gain a deeper understanding of the forces operating in recurrent situations and identify opportunities for leverage to benefit patients, staff, and healthcare organizations.

Facility's employment practices reflect affirmative action commitment.

The U.S. bishops' statements on racism over the last 40 years have addressed the structural as well as the personal aspects of racial discrimination. They have noted its persistence, described it as a moral and religious problem, and called for aggressive action among Catholics to combat it. "Meaningful equality" for blacks, however, remains a distant dream, the U.S. Supreme Court noted in a recent case involving institutional discrimination, because of the basically disadvantaged position into which blacks are born. As statistics on health, education, work, and standard of living illustrate, the socioeconomic opportunities available to blacks are markedly lower than those available to white Americans. Despite these odds, efforts to improve blacks' chances of contributing to society--through affirmative action programs--have achieved striking results in industry. Hospitals, as major workplaces, likewise should focus greater attention on developing and extending minority employment and advancement programs. Catholic health care facilities especially should evaluate their commitments to affirmative action. Increased efforts to eliminate discrimination in all aspects of a hospital's relationships with the business community as well as with its own employees will cause them to be recognized as symbols of justice not only in the workplace but also in society.

A community health education program.

Physicians, health educators, businessmen, and concerned citizens of Minnesota have developed a program for increasing community awareness of various aspects of healthy living. This program, based on collaborative effort, has been well received and well attended in the three rural communities in which it already has been presented. Most important of all, it has proven to be valuable for promoting individual responsibility for health.

Advanced practice roles in the managed care environment.

The role of the advanced practice nurse is based on expert clinical knowledge and skill and is practiced in multiple settings. As healthcare reform emerges, the context in which healthcare is delivered changes. The authors describe a creative approach to packaging and marketing the services of advanced practice nurses to the customers of the managed care system.

Identification of a transcription factor which interacts with the distal domain of the adenovirus IVa2 promoter.

For efficient in vitro transcription from the adenovirus IVa2 promoter, two upstream control regions, one proximal to the RNA initiation site at nucleotide position (np) -39 to -48 and the other a distal domain between np -152 and -179, are necessary. By using the band competition assay of Strauss and Varshavsky (Cell 37:889, 1984), we identified a factor which binds to the IVa2 promoter. Competition studies with various deletion mutants demonstrated that the region present 152 to 160 base pairs upstream of the IVa2 transcription start site was necessary for binding of this factor. DNase I footprinting assays demonstrated directly that this factor interacted with the distal domain of the IVa2 promoter. This factor was necessary for efficient transcription from the IVa2 promoter, as evidenced by the ability of a DNA fragment containing the binding site for this factor to inhibit transcription. Its role in bidirectional activation of transcription from major late and IVa2 promoters is discussed.

Preferential stimulation of transcription from simian virus 40 late and adeno IVa2 promoters in a HeLa cell extract.

Transcription of certain SV40 and adenovirus genes can be selectively enhanced under suitable in vitro assay conditions. The major SV40 late promoters and the adeno IVa2 promoter do not have TATA box sequences upstream from the start sites of transcription. Analyses of transcripts synthesized in vitro from the SV40 late promoters by "run-off" and S1 nuclease mapping methods show that when transcription takes place in the presence of 15 mM (NH4)2SO4, there is a 3- to 5-fold stimulation of RNA synthesis initiating from the late promoter at map position 185 and from the SV40 early promoters. RNA initiating from map position 325 is stimulated 40-fold under these conditions. When the (NH4)2SO4 concentration is increased to 50 mM (NH4)2SO4, there is a 90% suppression of transcription. Ammonium sulfate also enhances transcription from the adeno IVa2 promoter, which is not expressed in extracts that lack (NH4)2SO4 (Fire, A., Baker, C. C., Manley, J. L., Ziff, E. B., and Sharp, P. A. (1981) J. Virol. 40, 703-719; Lee, D. C., and Roeder, R. G. (1981) Mol. Cell. Biol. 1, 635-651). These modified assay conditions may allow detection of new classes of promoters.

Proximal and distal domains that control in vitro transcription of the adenovirus IVa2 gene.

The adenovirus IVa2 gene, which is expressed at an intermediate time in the viral infectious cycle, is separated from the adenovirus major late promoter (MLP) 5' start site by 210 base pairs and is transcribed from the opposite strand. In contrast to the MLP, the IVa2 gene does not contain a "TATA" box upstream from its 5' start sites. By using a series of deletion mutants, two upstream control regions that are rich in cytidine residues, one proximal to the cap site at nucleotide positions -39 to -48 and a distal domain between nucleotide positions -152 and -242 have been identified as essential for IVa2 transcription (IVa2 cap site is nucleotide position + 1). Transcription efficiency is decreased by 70-90% after the deletion of a proximal C-rich domain when either linear or supercoiled DNAs were used as template. However, distal sequences functioned as transcriptional control domains only with covalently closed DNA templates. The deletion of both the proximal and distal regions from covalently closed DNA templates reduces the levels of IVa2 transcription by a factor of 100-150. When the plasmid pAd242 that contains the 5' start sites of adenovirus MLP and IVa2 is transcribed, there is essentially a complete suppression of transcription of the adenovirus IVa2 gene. The transcription efficiency of IVa2 is increased 10-fold after deletion of the MLP cap site. A model based on a shared entry site for RNA polymerase II and competition between major late and IVa2 promoters is proposed to explain the in vitro transcriptional results.

Positive and negative control sequences within the distal domain of the adenovirus IVa2 promoter overlap with the major late promoter.

The RNA initiation sites of the adenovirus IVa2 and major late promoters (MLP) are separated by 210 base pairs and transcribed from opposite DNA strands. We had previously shown that they contained overlapping promoter sequences (V. Natarajan, M. J. Madden, and N. P. Salzman, Proc. Natl. Acad. Sci. U.S.A. 81:6290-6294, 1984). The transcription efficiencies of these two promoters were studied in vitro with templates of covalently closed circular DNAs that contained various deletion and point mutants. The distal control region of the IVa2 promoter that is located at nucleotide position (np) -152 to -242 from the RNA initiation site consists of at least two domains. The first distal domain, present between np -152 and -179, is necessary for efficient transcription of the IVa2 promoter, and it overlaps with sequences that have been shown to be necessary for efficient transcription of MLP. This region may serve as the entry site for the transcription machinery. The second distal domain consists of sequences present between np -211 and -242. These sequences are contained at the 5' end in the MLP transcript, and they inhibit transcription from the IVa2 promoter. However, these sequences are not necessary for transcription of the MLP with a covalently closed template but are needed for transcription with a linear template. The TATA box that is located at np -180 to -186 between these two domains has a critical role for efficient transcription of the MLP. A point mutation that reduces transcription from MLP by more than 80% stimulates transcription from IVa2 promoter by 10-fold. This finding is consistent with the proposal that MLP and IVa2 promoters share an entry site for transcription machinery and compete for its use.

Development needs of advance practice nurses in a managed care environment.

The authors describe an organizational change process driven by a critical cost containment effort in a teaching hospital in the northeastern United States. This process resulted in a major shift in the role of the clinical nurse specialist. Development priorities of clinical nurse specialists before and after redesign of the role are described.

A Y-box consensus sequence is required for basal expression of the human multidrug resistance (mdr1) gene.

Basal transcription of the human multidrug resistance (mdr1) promoter was studied by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in two parental and doxorubicin-resistant human tumor cell lines. Deletion of mdr1 DNA sequences to -89 relative to the start of transcription (at +1) had little effect on expression. Deletion of nucleotide sequences from -89 to -70, however, resulted in a 5-10-fold reduction in mdrCAT expression. DNase I footprint analysis demonstrated that the region from -85 to -70 was protected from nuclease digestion using nuclear extracts from these cell lines. The sequence between -82 and -73 is perfectly homologous with the 10-base pair Y-box consensus sequence found in the promoters of all major histocompatibility complex class-II (MHC II) genes. The Y-box sequence in MHC II genes is required for accurate and efficient transcription and contains the sequence CCAAT in the reverse orientation (Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C., and Mathis, D. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 6249-6253). Mutations in the reverse CCAAT sequence of the Y-box consensus substantially reduced expression of an mdrCAT vector and eliminated nucleoprotein binding in an electrophoretic mobility shift assay. These results suggest that proteins which bind to the putative Y-box consensus sequence are critical for basal transcriptional regulation of the human mdr1 gene.

Reversible transcriptional activation of mdr1 by sodium butyrate treatment of human colon cancer cells.

We investigated the mechanism of sodium butyrate (NaB)-mediated induction of mdr1 mRNA in parental (wild type) and multidrug-resistant (Ad1000) SW620 colon cancer cell lines. NaB treatment resulted in reversible, time-dependent increases in nuclear run-on transcription of endogenous mdr1 in these cell lines that paralleled the reversible increases of mdr1 mRNA in both timing and magnitude. In contrast, NaB treatment had no effect on mdr1 mRNA stability. Thus, the effects of NaB on mdr1 mRNA levels are fully attributable to altered mdr1 transcription. Furthermore, NaB induces the expression of transiently transfected chloramphenicol acetyltransferase reporter plasmids that are under the transcriptional control of the mdr1 promoter (mdrCAT vectors). Transfections using mdrCAT vectors modified by deletion and site-directed mutagenesis of the mdr1 promoter indicate that NaB-mediated induction of these vectors is at least partially dependent upon sequences present in the basal mdr1 promoter between -89 and +11 relative to the start site of transcription. The Y-box motif located between -82 and -73 contributes to NaB inducibility of mdrCAT vector expression in Ad1000 SW620 cells.

Identification of 5' and 3' sequences involved in the regulation of transcription of the human mdr1 gene in vivo.

The human mdr1 gene encodes a putative drug efflux pump (P-glycoprotein) whose overexpression is associated with the development of multidrug resistance (MDR). The promoter and 5'-flanking DNA of this gene were isolated from a human genomic DNA library and used to prepare a series of chloramphenicol acetyltransferase (CAT) fusion vectors under the transcriptional control of the mdr1 promoter (mdrCAT vectors). Transient transfection of these mdrCAT vectors produced CAT activities similar to those produced by transfection of CAT vectors containing viral promoters. The regulation of mdr1 expression was examined in two MDR tumor cell lines selected for resistance to doxorubicin and their corresponding parental cell lines. Although nuclear run-on analysis indicates that the expression of the mdr1 gene in these two MDR cell lines is regulated by transcriptional mechanisms, mdrCAT expression was not significantly increased in either of these lines relative to parental cells. Thus, the sequences involved in the transcriptional regulation in these cells are apparently not included in the constructs studied (-4741 to +286). Analyses of a series of deletion constructs show that the basal mdr1 promoter activity is encoded by sequences that span a region adjacent to the transcription start site (-134 to +286) and that sequences 3' to the start of mdr1 transcription are necessary for proper initiation of transcription in vivo. Structural and functional studies indicate that an initiator (Inr) sequence surrounding the major transcription start site governs accurate initiation of mdr1 transcription.

Persistent herpes simplex virus infections established in two Burkitt lymphoma derived cell lines.

Examination of P3HR-I cells (Epstein-Barr virus [EBV] producer) persistently infected with the MAL strain of herpes simplex virus type I (HSV-I) suggested that only a few cells were actively producing a virus indistinguishable from HSV-I (MAL) despite the presence of immunofluorescent HSV-I antigens associated with the majority of cells. EBV-specific immunofluorescence was not altered in HSV-I persistently infected P3HR-I cells. HSV-I persistently infected cells, labelled for 72 h with 14C-thymidine, incorporated approx. 8% of the label into cell associated HSV-I DNA as resolved by caesium chloride gradients. Values greater than 8% of the total were suggested by hybridization of gradient fractions with 3H-HSV-I DNA. To determine whether the establishment of HSV persistent infections in Burkitt lymphoma derived cells was a general phenomenon, six strains of HSV-I (MAL, KOS, Patton, Syn R, BF and SYN V) and two strains of type 2 (333 and MS) were used to infect the P3HR-I and Raji (EBV non-producer) cell lines derived from Burkitt lymphomas. In P3HR-I cells, persistent infections were established with all strains of HSV-I but not with HSV-2. In Raji cells, persistent infections were established with all strains of HSV-I, except Syn V, and with both strains of HSV-2. No external support was required to maintain these infections.