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PubMed1991

Characterization of the level, target sites and inheritance of cytosine methylation in tomato nuclear DNA.

The tomato nuclear genome was determined to have a G + C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G + C content even lower (32% average) whereas coding regions are considerably richer in G + C (46%). 5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development. Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI) are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco. The low G + C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mC----T. This is supported by the fact that G + C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions. Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.

Base Composition↗

PubMed1993

A transcription initiation site for the hepatitis B virus X gene is directed by the promoter-binding protein.

Recent studies have demonstrated the transacting function of the X gene product of hepatitis B virus. However, little information is available on the regulation of X gene expression. In this report, we first investigate a cellular factor regulating X gene transcription by DNA transfection, using the human hepatoma cell line HuH-7, which is permissive for HBV replication as well as X mRNA transcription. A sequence-specific cellular factor was found to bind to the promoter region upstream of the first ATG (nucleotide [nt] 1248) of the X open reading frame. DNase I footprinting analysis showed the binding sequence of this factor to be situated between nt 1097 and 1119, where an 8-bp palindrome structure resides. S1 nuclease analysis of X gene transcripts demonstrated the binding site to be adjacent to two major start sites (nt 1117 and 1125) of X mRNA. Second, we demonstrate that introduction of a mutation into the binding site gives rise to a loss of the binding with a concomitant shift of the transcription start site of X mRNA beyond the 8-bp palindrome structure, causing it to become more heterogeneous. Thus, the promoter-binding protein appears to be involved in directing the transcription initiation site of the X gene toward the downstream region of the X promoter when X protein is produced from X mRNA.

Base Sequence↗

PubMed1995

Functional analysis of the human pro-opiomelanocortin promoter in the small cell lung carcinoma cell line DMS-79.

DMS-79 is a human cell line derived from a small cell lung carcinoma (SCLC), which expresses the pro-opiomelanocortin (POMC) gene. We took it as a model in which to study the mechanism of POMC gene expression in these tumors: precursor processing is altered and gene expression is essentially unresponsive to glucocorticoids. POMC gene structure appeared normal by Southern blot analysis, indicating that gene rearrangement was not responsible for its expression in DMS-79. Indeed, using transient expression of human POMC-luciferase fusion genes in DMS-79, we showed that (1) the normal human POMC promoter was functional in DMS-79, and (2) the same proximal promoter region (-417; + 21) produced the full transcriptional activity in DMS-79 and in the mouse pituitary cell line AtT-20. Progressive 5' deletion analysis revealed differences between AtT-20 and DMS-79: region (-611; -376) was active in AtT-20 and not in DMS-79, whereas region (-95; -161) was active in both cell lines and (-376; -417) was only active in DMS-79. The latter partially overlaps a motif homologous to the DE-2 rat element which confers the tissue-specific expression of POMC in AtT-20 cells; however, this motif had no effect in DMS-79. These data suggest that POMC gene transcription is achieved through a different set of transacting factors in DMS-79 and AtT-20. Altogether, our results provide evidence that DMS-79 is a valid model of tumors responsible for the ectopic ACTH syndrome and that the mechanism of POMC gene expression in these SCLC cells is different from that in pituitary cells.

Animals↗

PubMedDate not supplied

Community benefits: how do for-profit and nonprofit hospitals measure up?

The rise of the for-profit hospital industry has opened a debate about the level of community benefits provided by non-profit hospitals. Do nonprofits provide enough community benefits to justify the community's commitment of resources to them, and the tax-exempt status they receive? If nonprofit hospitals convert to for-profit entities, would community benefits be lost in the transaction? This debate has highlighted the need to define and measure community benefits more clearly. In this Issue Brief, the authors develop a new method of identifying activities that qualify as community benefits, and propose a benchmark for the amount of benefit a nonprofit hospital should provide.

Benchmarking↗

PubMed2005

Phosphorylation of octamer-binding transcriptional factor may be correlated with H2B histone gene repression during 12-O-tetradecanoylphorbol 13-acetate-dependent differentiation of HL-60 cells.

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA), the binding pattern of nuclear proteins to various elements in the human H2B histone gene upstream region have been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA rapidly reduced at 24 h in TPA-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of TPA. In DNase I footprinting analysis, one nuclear factor (octamer-binding transcription factor, OTF) bound at -42 bp (octamer motif), before and after TPA-induced differentiation of HL-60 cells. One DNA-protein complex (OTF) was formed by DNA mobility shift assay when octamer element was incubated with nuclear extract of undifferentiated HL-60 cells. In DNA mobilith shift assay, OTF vanished, and phosphorylated OTF (p-OTF) newly appeared during TPA-induced differentiation. p-OTF was not detected after pretreatment of the protein kinase C inhibitor, staurosporin, but was not changed after CHX treatment. TPA-induced repression of H2B histone mRNA was also restored after pretreatment of staurosporin. These results suggest that OTF is phosphorylated by protein kinase C during TPA-induced differentiation of HL-60 and the transcriptional repression of the H2B histone gene may be mediated by protein kinase C-dependent phosphorylation of OTF.

Base Sequence↗

PubMed1997

The valuation of health care intangible assets.

Health care entities (and especially medical practices) are valued for a number of reasons: sale transaction pricing and structuring, merger formation and dissolution, taxation and regulatory compliance, and litigation support and dispute resolution. The identification and quantification of the entity's intangible assets are often the most important aspects of the valuation. This article illustrates the generally accepted methods for valuing health care-related intangible assets.

Costs and Cost Analysis↗

PubMed1995

Reduced level of octamer binding transcription factor (Oct-1) is correlated with H2B histone gene repression during differentiation of HL-60 cells by all-trans retinoic acid.

To gain insight on the role of transacting factors in the regulatory mechanism of H2B histone gene expression during the differentiation of HL-60 cells by all-trans retinoic acid (retinoic acid), the binding pattern of the nuclear proteins to various elements in the human H2B histone upstream region has been investigated with DNase I footprinting and DNA mobility shift assay. The level of H2B histone mRNA was markedly reduced at 48 hr in retinoic acid-treated HL-60 cells. The H2B histone mRNA was repressed in proportion to the concentration of retinoic acid. In DNase I footprinting analysis, a nuclear factor (octamer binding transcription factor, Oct-1) bound at -42 bp (ATTTGCAT) both before and after retinoic-acid-induced differentiation of HL-60 cells. One DNA-protein complex was formed by DNA mobility shift assay, and the level of Oct-1 decreased during retinoic-acid-induced differentiation. In the cycloheximide-treated HL-60 cells, the level of Oct-1 also reduced. These results suggest that the transcriptional repression of H2B histone gene during retinoic-acid-induced differentiation in HL-60 cells may be mediated by reduced level of Oct-1.

Base Sequence↗

PubMed2003

MADGE: scalable distributed data management software for cDNA microarrays.

MOTIVATION: The human genome project and the development of new high-throughput technologies have created unparalleled opportunities to study the mechanism of diseases, monitor the disease progression and evaluate effective therapies. Gene expression profiling is a critical tool to accomplish these goals. The use of nucleic acid microarrays to assess the gene expression of thousands of genes simultaneously has seen phenomenal growth over the past five years. Although commercial sources of microarrays exist, investigators wanting more flexibility in the genes represented on the array will turn to in-house production. The creation and use of cDNA microarrays is a complicated process that generates an enormous amount of information. Effective data management of this information is essential to efficiently access, analyze, troubleshoot and evaluate the microarray experiments. RESULTS: We have developed a distributable software package designed to track and store the various pieces of data generated by a cDNA microarray facility. This includes the clone collection storage data, annotation data, workflow queues, microarray data, data repositories, sample submission information, and project/investigator information. This application was designed using a 3-tier client server model. The data access layer (1st tier) contains the relational database system tuned to support a large number of transactions. The data services layer (2nd tier) is a distributed COM server with full database transaction support. The application layer (3rd tier) is an internet based user interface that contains both client and server side code for dynamic interactions with the user. AVAILABILITY: This software is freely available to academic institutions and non-profit organizations at http://www.genomics.mcg.edu/niddkbtc.

Database Management Systems↗

PubMed2004

Tools from the new institutional economics for reforming the delivery of veterinary services.

The new institutional economics offers a range of analytical tools and insights which can be used in restructuring the animal health services of a country. This paper discusses three of the most important--externalities, transaction costs and asymmetric (unequal) information. These tools can be used to craft a system which is sensitive to the particular institutional heritage of a country and to its political, social and economic realities. Institutions are important in this process and the best way for one society is often not the optimal choice for another.

Animals↗

PubMed2001

Transcriptional regulation of the p67phox gene: role of AP-1 in concert with myeloid-specific transcription factors.

We have investigated the myeloid-specific transcriptional regulation of p67(phox), an essential component of phagocyte respiratory burst NADPH oxidase. Analysis was carried out on the p67(phox) 5'-flanking region from -3669 to -4 (relative to ATG), including the first exon and intron and part of the second exon. The construct extending from -985 to -4 produced the highest luciferase activity in myeloid HL-60 cells but was not active in HeLa or Jurkat cells, indicating myeloid-specific expression. Four active elements were identified: Sp1/Sp3 at -694, PU.1 at -289, AP-1 at -210, and PU.1/HAF1 at -182, the latter three being in the first intron. These cis elements bound their cognate transacting factors both in vitro and in vivo. Mutation of the Sp1, PU.1, or PU.1/HAF1 site each decreased promoter activity by 35-50%. Mutations in all three sites reduced promoter activity by 90%. However, mutation of the AP-1 site alone nearly abolished promoter activity. The AP-1 site bound Jun and Fos proteins from HL-60 cell nuclear extract. Co-expression with Jun B in AP-1-deficient cells increased promoter activity by 3-fold. These data show that full p67(phox) promoter activity requires cooperation between myeloid-specific and nonmyeloid transcription factors, with AP-1 being the most critical for function.

Base Sequence↗

PubMed2000

[Chronological analyses of neuropathological and molecular biological changes in affected spinal cord of HTLV-I-infected rat (HAM rat disease)].

The author chronologically analyzed neuropathological aspects of the demyelination process in spinal cords of a rat model of human T-cell leukemia virus type I (HTLV-I)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) from early asymptomatic to late disease stage for clarifying the pathogenetic roles of HTLV-I in central nervous system. There was no significant difference in histopathological and immunohistochemical findings between the rats within 7 months after the HTLV-I infection and age-matched controls. The first sign of demyelination was the appearance of apoptotic cell death beginning at 7 months after the infection and the apoptotic cell number gradually increased to 12 cells per a whole horizontal section of the spinal cord, in contrast to 5 cells in the control rats. The majority of the apoptotic cells were shown to be oligodendrocytes by immunohistochemical stain with an anti-myelin oligodendrocyte glycoprotein antibody. Increment of activated microglia/macrophages started at 9 months after the infection and they rapidly increased from 15 months to reach 600 cells per a whole horizontal section, in contrast to 300 cells in the control rats. Rapid increase of gemistocytic astrocytes was found from 20 months after the infection (the late disease stage). Molecular analysis of the spinal cords revealed that HTLV-I provirus DNA was evident as early as 4 months after the infection, and massages of HTLV-I pX and tumor necrosis factor (TNF)-alpha began to be expressed at 7 months, just before or at the same time as the appearance of the apoptotic cells. The collective evidence suggests that the apoptotic death of oligodendrocytes, which may be induced either directly by the local expression of HTLV-I or indirectly by upregulated cytotoxic humoral mediators, such as TNF-alpha, through the transactive function of p40 Tax, is the major cause of chronic progressive myelopathy in WKAH rats with HTLV-I infection.

Animals↗

PubMed1998

Competition as a means of procuring public services. Lessons for the UK from the US experience.

Competition is now widely used as the means of choosing the providers of essential public services in the USA and the UK. Many different approaches are found in the USA and there are useful lessons for the UK. With particular reference to mental health and substance abuse services, describes the effects of using competitive tendering on users, providers, purchasers and citizens and examines the problems of specification, transaction costs, the use of consultants, supply, the level playing field, trust, innovation, local accessibility and accountability. Ends with discussion of co-operation and collaboration and the emergence of monopolies and integrated delivery systems in the USA and concludes by finding politics and political decision making of overriding importance.

Attitude of Health Personnel↗

PubMed1995

The hospital information system as a source for the planning and feed-back of specialized health care.

1. INTRODUCTION. In university hospitals, choices are made to which extend specialized health care will be supported. It is characteristic, for this type of care, that it takes place in a process of the continual advance of medical technology and the growing awareness by consumers and payors. Specialized healthcare contributes to the hospital qualifiers having a political and strategic impact. The hospital board needs information for planning and budgeting these new tasks. Much of the information will be based on data stored in the Hospital Information System (HIS). Due to load limitations, instant retrieval is not preferred. A separate executive information system, uploaded with HIS data, features statistics, on a corporate level, with the power to drill-down to detailed levels. However, the ability to supply information on new types of healthcare is limited since most of these topics require a flexible system for new dedicated cross-sections, like medical treatment from several specialisms and functional levels. 2. DATA RETRIEVAL AND DISTRIBUTION. During the information analysis, details were gathered on the necessary working procedures and the administrative organization, including the data registration in the HIS. In the next phase, all relevant data was organized in a relational datamodel. For each topic of care, dedicated views were developed at both low and high aggregation levels. It revealed that a matching change of the administrative organization was required, with an emphasis on financial registration aspects. For the selection of relevant data, a bottom-up approach was applied, which was based on the registrations starting from the patient administrative subsystem, through several transactional systems, ending at the general ledger in the HIS. Data on all levels was gathered, resulting in medical details presented in quantities, up to financial figures expressed in amounts of money. This procedure distinguishes from the predefined top-down techniques generally used for management and executive information systems. Data was regularly collected from the HIS, then converted and reorganized into relational datasets using XBase protocols. After having performed central quality controls and privacy protection measures, the datasets were distributed electronically to local PCs. Standard low-cost software packages enable analyses by user-friendly selection and presentation facilities. 3. EVALUATION. The method developed for data retrieval is flexible and easy to implement. If all basic data is registered in the HIS, the procedure can be applied for all strategic hospital functions that require planning and controlling during a certain time. Critical success factors and pitfalls will be presented in the poster. Using one consistent dataset, the information required about production and budget is presented at several functional levels and is quantified in units familiar to that level. The motivation for fast and accurate registration in the HIS was improved from the moment the medical and administrative staff recognized their own data in the feed-back on specialized health care.

Decision Making, Organizational↗

PubMed2006

Effects of pelvic, pudendal, or hypogastric nerve cuts on Fos induction in the rat brain following vaginocervical stimulation.

In the female rat, genitosensory input is conveyed to the central nervous system predominantly through the pelvic, pudendal, and hypogastric nerves. The present study examined the relative contribution of those three nerves in the expression of Fos immunoreactivity within brain regions previously shown to be activated by vaginocervical stimulation (VCS). Bilateral transection of those nerves, or sham neurectomy, was conducted in separate groups of ovariectomized, sexually-experienced females. After recovery, females were primed with estrogen and progesterone and given either 50 manual VCSs with a lubricated glass rod over the course of 1 h. VCS increased the number of neurons expressing Fos immunoreactivity in the medial preoptic area, lateral septum, bed nucleus of the stria terminalis, ventromedial hypothalamus, and medial amygdala of sham neurectomized females. Transection of the pelvic nerve reduced Fos immunoreactivity in the medial preoptic area, bed nucleus of the stria terminalis, ventromedial hypothalamus, and medial amygdala, whereas transection of the pudendal nerve had no effect. In contrast, transection of the hypogastric nerve increased Fos immunoreactivity in the medial preoptic area and lateral septum, whereas transaction of the pelvic nerve increased Fos immunoreactivity in the lateral septum, following VCS. All females given VCS, except those with pelvic neurectomy, displayed a characteristic immobility during each application. These data confirm that the pelvic nerve is largely responsible for the neural and behavioral effects of VCS, and support a separate function for the hypogastric nerve.

Analysis of Variance↗

PubMed1991

Communication about dental health in Norwegian adults.

The present study describes the amount and distribution of communication on dental health issues in Norwegian adults. The empirical data stem from a representative sample of Norwegians aged 15 and above, and were collected by means of personal interviews by the poll organization Norges Markedsdata in 1983. Edentulous subjects were excluded, and the study group finally comprised 1225 subjects. The dependent variable - dental communication - was a dichotomy based upon whether or not the respondents had communicated with friends about dental health during the last 6 months. The following independent variables were used: age, sex, marital status, education, use of dental services, number of teeth, knowledge of prevention, information about dental health from dentists and media, use of dental floss and toothpicks. The empirical analysis was performed using contingency table analysis and Multiple Classification Analysis (MCA). Separate analyses were performed for men and women. Significantly more women (27.6%) than men (14.5%) reported dental communication. In addition, the model provided a better fit to the data on women compared to men in terms of R2 (12.5% and 8.9%). These observations can to some extent be accounted for by the existing sex-role pattern. The only predictor which showed a consistent direct effect across sex was whether the respondents had received information about dental health from media. This means that reference to the interpersonal context of mass communication perhaps best describes the complex transactional interplay of media and interpersonal sources in dental health matters.

Adolescent↗

PubMed1996

Diversity of Trypanosoma cruzi stocks and clones derived from Chagas disease patients. II. Isozyme and RFLP characterizations.

Isoenzyme and RFLP analyses were carried on freshly isolated Trypanosoma cruzi stocks and subsequent clones derived from patients with chronic Chagas disease. The isoenzymes separated the parasite stocks and clones in two groups: The stock hSLU239 (group I), isolated from a heart disease patient, showed the zymodeme 3 (Z3) profile (M. A. Miles et al., 1977, Transactions of the Royal Society of Tropical Medicine and Hygiene 71, 217-225). The stock mSLU142 (group II), isolated from a digestive disease (megaesophagus) patient, showed the Z2 profile. The parasite clones m1, m2, m3, and m4, derived from mSLU142, and clones h1 and h2, derived from hSLU239, showed isoenzyme profiles similar to those of Z2 and ZA (Miles et al. 1977; J. A. Romanha, 1982, Thesis, Universidade Federal de Minas Gerais). Furthermore, the T. cruzi clones derived from the cardiac disease patient differed from those derived from the megacolon patient in 3 of the 13 enzymes analyzed. RFLP analysis showed polymorphism at the EcoRI and PstI restriction fragments of the DNA sequences coding the glycolytic enzymes ALD, GPI, GAPDH, and PYK and separated the T. cruzi stocks and clones in three groups: I, comprising the stock hSLU239 and clone m4, which was classified as homozygous CC, BB, AA, and AA for the ALD, GPI, PYK, and GAPDH genes, respectively; II, formed by the parasite stock mSLU142 and clones h1 and h2 (derived from hSLU239), which was classified as homozygous AA, AA, CC, and BB for ALD, GPI, PYK, and GAPDH genes, respectively . These findings show that the infection of each Chagas disease patient may be produced by genetically diverse mixed parasite populations.

Animals↗

PubMed1985

Reforming Medicare provider payment.

The basic thesis of this paper is that, in the short to medium term (that is to say, the politically relevant term), fee-for-service payment will be the dominant form of Medicare transaction--and that is not such a bad thing. Capitated arrangements will grow, and should be encouraged, especially for long-term-care services, but for reasons to be discussed, it would be a mistake to put too many policy eggs into that basket. As the nascent Prospective Payment System (PPS) for hospital care reveals, fee-for-service payment can take almost an infinite variety of forms, and the connection between the choice of form and the cost impact is highly variable and somewhat uncertain. PPS needs considerable improvement; so, unquestionably, does the current nonsystem of physician payment. An analysis of potential improvements in both arenas will constitute the bulk of this discussion. That emphasis reflects not only the personal predilections of the author, but also the contention that those are the most sensible things to talk about.

Aged↗

PubMed2000

Comparative analysis of translation efficiencies of hepatitis C virus 5' untranslated regions among intraindividual quasispecies present in chronic infection: opposite behaviors depending on cell type.

Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5' untranslated region (5' UTR). While exhibiting the most highly conserved sequence within the genome, the 5' UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5' UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

5' Untranslated Regions↗