An operational model for patient-centered informatics.

There are no multidisciplinary operational models to guide nursing informaticists and clinical system users in the design and implementation of computer-supported multidisciplinary care. The Patient-Centered Informatics Model is offered as just such a pragmatic guide. It fuses earlier work with new concepts and allows a visual depiction of crucial elements-influencing factors such as regulations and healthcare delivery models, system attributes such as healthcare delivery methods, knowledge base and supporting technology and categories of results of application processing. The model can help users and executives organize their thinking about the design, implementation, and evaluation of clinical systems in complex settings.

The activated mammary gland specific nuclear factor (MGF) enhances in vitro transcription of the beta-casein gene promoter.

The hormonal induction of the beta-casein gene in mammary epithelial cells is dependent on the action of peptide and steroid hormones. Epidermal growth factor, insulin, glucocorticoids and prolactin act in a sequential manner to regulate the transcription of the gene. We have studied the hormonal requirements as well as the nuclear proteins which are involved in the induction process. In vitro transcription in cell free nuclear extracts has been used to demonstrate the central role of the mammary gland specific nuclear factor, MGF, in the mediation of the hormonal signals to the transcription machinery. A gene construct comprising 344 nucleotides of wild type beta-casein promoter sequence and a G-free cassette of 220 nucleotides was used to test the activity of nuclear extracts in the in vitro transcription experiments. A construct in which the proximal MGF binding site in the beta-casein promoter region has been inactivated by mutation and a construct regulated by the adenovirus major late promoter served as controls. Nuclear extracts were prepared from Sf insect cells, HeLa cells and mammary epithelial cells of lactating rats. Strong transcription of the wild type beta-casein promoter construct was observed in the mammary cell extract, weak transcription in the extracts of Sf insect cells and the HeLa cells. The mutation of the MGF binding site drastically reduced the in vitro transcription in the mammary gland cell extract. Beta-casein promoter activity was also compared in nuclear extracts from uninduced and lactogenic hormone induced HC11 mammary epithelial cells. Extracts from induced cells are more efficient in the support of beta-casein gene transcription.

The effect of point of care technology on the quality of patient care.

It is thought that new technologies like computers at the patient's bedside, or point of care technology (PCT) improve nursing productivity, documentation, patient satisfaction and decrease costs. Using the Health Care Technology Assessment (HCTA) framework, (safety, cost, effectiveness, social impact), a descriptive and quasi-experimental study was performed to test the effectiveness and explain the social impact of PCT. A sample of 90 patients from five nursing units in three hospitals were obtained for the study. Half of the patients had computers at their bedside. Data were collected on a hospital pretest/posttest unit and two comparison and experimental units. The main null hypothesis was: There is no difference in the quality of patient care on nursing units with and without PCT. Quality of patient care was measured by patient satisfaction and a nursing care documentation instruments. This hypothesis was rejected. While patients were generally very satisfied with their nursing care on all units, when controlling for time and the presence of the computer, patients who did not have PCT were more satisfied than patients in rooms with PCT. Furthermore, the charts of patients with PCT were less compliant to documentation standards. Conversely, a sub sample of these same patients expressed positive responses to the bedside computer and technologies in their room and this concurred with the current literature. The benefits of the technology were found to outweigh the costs of PCT from the literature review. There was not enough in the literature to draw conclusions about the safety of PCT. In summary, the quality of patient care did not improve with the implementation of PCT in this study.(ABSTRACT TRUNCATED AT 250 WORDS)

Ha-ras and v-raf oncogenes, but not int-2 and c-myc, interfere with the lactogenic hormone dependent activation of the mammary gland specific transcription factor.

beta-Casein milk protein expression is regulated synergistically by the lactogenic hormones dexamethasone, insulin, and prolactin. This regulation has been observed in vivo and in the mammary epithelial cell line HC11. An important mediator of hormone action on beta-casein gene transcription is the mammary gland specific transcription factor MGF. Epidermal growth factor is a potent growth promoter for HC11 cells and causes a state of predifferentiation in confluent HC11 cells. Epidermal growth factor receptor activation, however, antagonizes the effects of the lactogenic hormones on MGF activation and beta-casein gene transcription during the induction of predifferentiated cells. We examined the effects of two downstream components of the epidermal growth factor receptor mitogenic signaling pathway on MGF and beta-casein promoter activity. Constitutively activated Ha-ras or v-raf oncogenes were introduced into HC11 cells. HC11 cell lines transfected with Ha-ras or v-raf oncogenes assumed transformed properties and were blocked in the lactogenic induction of MGF and beta-casein gene transcription. This indicates that the same components instrumental in the mitogenic pathway are also involved in the block of differentiation specific gene expression in HC11 cells. This block is not due to the transformed state of the HC11 cells. Introduction and high expression of the int-2 gene, a member of the fibroblast growth factor gene family, does not interfere with MGF and beta-casein induction. Overexpression of the c-myc gene, causing mammary epithelial cell transformation, also does not suppress MGF activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammary gland-specific nuclear factor activity is positively regulated by lactogenic hormones and negatively by milk stasis.

The mammary gland-specific nuclear factor (MGF) is a crucial contributor to the regulation of transcription from the beta-casein gene promoter. The beta-casein gene encodes a major milk protein, which is expressed in mammary epithelial cells during lactation and can be induced by lactogenic hormones in the clonal mammary epithelial cell line HC11. We have investigated the specific DNA-binding activity of MGF in mammary epithelial cells in vivo and in vitro. Comparison of MGF in HC11 cells and mammary gland cells from lactating mice revealed molecules with identical DNA-binding properties. Bandshift and UV cross-linking experiments indicated that MGF in HC11 cells has a higher mol wt than MGF found in mice. Little MGF activity was detected in nuclear extracts from HC11 cells cultured in the absence of lactogenic hormones. Lactogenic hormone treatment of HC11 cells led to a strong induction of MGF activity. The induction of MGF activity as well as utilization of the beta-casein promoter were suppressed when epidermal growth factor was present in the tissue culture medium simultaneously with the lactogenic hormones. In lactating animals, MGF activity is regulated by suckling, milk stasis, and systemic hormone signals. The mammary glands from maximally lactating animals, 16 days postpartum, contain drastically reduced MGF activity after removal of the pups for only 8 h. The down-regulation of MGF by pup withdrawal was slower in early lactation, 6 days postpartum. We also investigated the relative contributions of local signals, generated by milk stasis, and systemic hormone signals to the regulation of MGF activity. The access to one row of mammary glands of lactating mothers was denied to the pups for 24 h. High levels of MGF were found in the accessible mammary glands, and intermediate levels of MGF were found in the inaccessible glands of the same mouse. Very low MGF levels were detected when the pups were removed from the dams for 24 h. We conclude that systemic as well as local signals cooperate in the in vitro regulation of MGF activity.

Developmental and environmental regulation of a mammary gland-specific nuclear factor essential for transcription of the gene encoding beta-casein.

During the lactation period, mammary epithelial cells secrete large amounts of milk proteins. The coordinate regulation of milk protein expression is effected by the lactogenic hormones. We have investigated the activity of a mammary gland-specific transcription factor (MGF), which mediates hormonal influences at the level of a milk protein gene promoter. MGF-binding sites are present in the promoters of the most abundantly expressed milk protein genes. Mutation of the MGF-binding site in the beta-casein gene promoter completely abolishes responsiveness of the promoter to lactogenic hormones in cultured mammary epithelial cells. MGF activity is closely controlled in vivo. High MGF levels were found in mouse mammary gland nuclear extracts toward the end of pregnancy and during lactation. Withdrawal of suckling pups from their mothers during the lactation period caused a strong and rapid decrease of MGF activity. Readdition of pups to their mothers restored maximal MGF levels within 4 hr. We investigated MGF phosphorylation as a possible posttranslational modification responsible for regulation of the DNA-binding activity of MGF. Treatment of nuclear extracts from lactating mammary glands with potato acid phosphatase abolished MGF-binding activity. Casein kinase II phosphorylation of nuclear extracts from animals withdrawn from their pups for 24 hr enhanced MGF-binding activity. These results suggest that the reversible activation of MGF by suckling and withdrawal might be mediated by the action of kinases and phosphatases.

Proteins encoded in the 81.2- to 85.0-map-unit fragment of Autographa californica nuclear polyhedrosis virus DNA can be translated in vitro and in Spodoptera frugiperda cells.

We have previously demonstrated that five open reading frames exist in the nucleotide sequence of the 81.2- to 85.0-map-unit (m.u.) segment of plaque isolate E of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The corresponding polypeptides are 9.8, 12.1, 36.6, 25.0, and 48.2 kDa in size (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 63:1494, 1989), and we have investigated whether these proteins can be translated in infected cells. On subfragments of this viral DNA segment, mRNAs were selected from AcNPV-infected Spodoptera frugiperda insect cells at different times postinfection (p.i.). The in vitro translation of these RNAs in a rabbit reticulocyte-derived cell-free translation system yielded polypeptides of approximately 10 to 11, 12 to 14, 28, 36 to 38, and 48 to 50-kDa which were commensurate in size with the theoretically expected values. mRNAs for the 28- and 48- to 50-kDa proteins were identified by their translation products at 6 h p.i., and mRNAs for the 10- to 11-, 12- to 14-, and 36- to 38-kDa proteins were identified by their translation products at 12 h p.i. We constructed an AcNPV recombinant which carried in its polyhedrin gene the 3.9-kbp EcoRI-HindIII (81.8 to 84.8 m.u.) subfragment of the EcoRI J segment. Nucleotide sequence determinations revealed that the intact polyhedrin promoter lay adjacent to the additional 81.8- to 84.8-m.u. fragment in this recombinant. In S. frugiperda cells, which were infected with the recombinant AcNPV, a protein of 36 to 38 kDa was detected at 44 h p.i. in larger amounts than after infection with the nonrecombinant virus. However, there was no evidence for larger amounts of RNA derived from the 81.8- to 84.8-m.u. fragment in recombinant-infected cells. Recombinant-infected cells lacked the polyhedrin polypeptide. The synthesis of the 36- to 38-kDa polypeptide in recombinant- or AcNPV-E-infected S. frugiperda cells could be demonstrated by immunoprecipitation experiments. Peculiarly, this polypeptide was present in the cytoplasm as a 64-kDa glycoprotein. These data corroborate the notion that at least some of the open reading frames encoded in the 81.2- to 85.0-m.u. segment of AcNPV can be expressed in S. frugiperda cells.

The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells.

This report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The following studies have been summarized. (1) On the mode of transcription of the AcNPV genome in insect cells. (2) Translation of proteins encoded in the 81.2 to 85.0 map unit segment of AcNPV. (3) Inserts of insect cell DNA in the AcNPV genome. (4) Expression of influenza (fowl plague) virus haemagglutinin in Spodoptera frugiperda insect cells, and successful immunization of chickens. (5) Synthesis of the influenza virus haemagglutinin in insect larvae by recombinant AcNPV. This insect virus system will continue to serve as a model for research on the molecular biology of insects. Moreover, the baculovirus system has been recognized as a very efficient and safe eukaryotic expression vector.

Overlapping sets of viral RNAs reflect the array of polypeptides in the EcoRI J and N fragments (map positions 81.2 to 85.0) of the Autographa californica nuclear polyhedrosis virus genome.

In several parts of the Autographa californica nuclear polyhedrosis virus (AcNPV) genome, nested sets of overlapping RNAs with common 3' or 5' termini have been recognized. In the present report, the pattern of viral transcription and the arrangement of viral gene products in the region of 81.2 to 85.0 map units were investigated. In this segment of the AcNPV genome, at least nine size classes of viral RNA were identified which ranged in size from 1.3 kilobases (kb) to 4.6 kb and exhibited common 3' termini. The detailed restriction map and the nucleotide sequence of this part of the AcNPV genome were determined. Computer analyses revealed several open reading frames (ORFs) on the rightward-transcribed strand with potential TATA and CAAT signals preceding many of the potential ORFs and the 5' termini of some of the mapped RNAs. The leftward-transcribed strand was devoid of major ORFs. The presumptive polypeptides encoded by the larger ORFs ranged in size from 11.3 to 55.6 kilodaltons (kDa). The amino acid sequence of the presumptive polypeptide encoded by ORF3, a 33.6-kDa molecule, exhibited an unusual, clustered 16-fold repeat of the dipeptide arginine-serine in a protein that showed an overall preponderance of basic amino acids. The results of in vitro translation experiments with hybrid-selected RNAs homologous to internal subfragments of the 81.2- to 85.0-map-unit region yielded polypeptides of approximately 28, 34 to 36, and 48 to 50 kDa, which were close in size to the lengths of the major ORFs derived from the nucleotide sequence. The localizations of individual size classes of RNAs in the 81.2- to 85.0-map-unit region of the viral genome were determined precisely at the 3' and 5' termini by S1 protection analyses. Within a sequence of eight nucleotides, all RNAs had the same 3' terminus, which lay close to multiple polyadenylation signals. The initiation sites of the nine different RNA size classes were precisely mapped. As the cap sites of the smaller RNAs (less than 1.8 kb) were determined by S1 protection analyses, a multitude of RNA initiation sites became apparent. It was also shown that the different RNA size classes in the 81.2- to 85.0-map-unit region were detectable as early as 2 h and at least until 36 to 48 h after infection. In unselected cytoplasmic RNA, the size classes of viral RNAs specific for the EcoRI J fragment were detectable early as well as late after infection, although at early times the larger RNAs were detectable in smaller amounts.(ABSTRACT TRUNCATED AT 400 WORDS)

Lipases and lipids in human milk: effect of freeze-thawing and storage.

Frozen storage is often used by milk banks to preserve expressed human milk for later use. Optimal storage and handling conditions which ensure minimum alteration of lipid composition have not been well defined. Therefore we investigated the effect of rapid freeze-thawing and storage conditions (-20 and -70 degrees C) on the free fatty acid (FFA) levels and on the activities of lipoprotein lipase (LPL) and bile salt-stimulated lipase (BSSL) in human milk. Since during mechanical expression leakage of serum components into milk may occur, we also investigated the effect of the presence of serum on human milk LPL during storage. Lipase activity levels were unaffected by rapid freeze-thawing (x3) followed by storage for 1 month at -20 or -70 degrees C. LPL activity (nmol FFA released/ml milk/min) was 414 +/- 128, 451 +/- 37, and 351 +/- 20 and BSSL activity (mumol FFA/ml milk/min) was 5.7 +/- 0.7, 5.5 +/- 0.8, and 5.7 +/- 0.2 in fresh, freeze-thawed, and stored milk, respectively. FFA levels (% of total lipid) were 3.01 +/- 1.05 and 10.3 +/- 1.6 in fresh-frozen milk stored at -70 and -20 degrees C for 5 months, and 3.78 +/- 1.08 and 13.60 +/- 1.25 in specimens of freeze-thawed (x3) before storage at -70 or -20 degrees C. Addition of serum had no effect on milk LPL at either temperature. We conclude that LPL and BSSL remain fully active during frozen storage of human milk and that milk fat is hydrolyzed at -20 degrees C but not at -70 degrees C. We suggest that banked human milk be stored routinely at -70 degrees C.