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PubMed2006

Coaxial multi-electrode cell ('O-trap') for high-sensitivity detection at a multiple frequency in Fourier transform ion cyclotron resonance mass spectrometry: main design and modeling results.

Separation of the functions of ion excitation and detection between different cell compartments allows for implementation of excitation and detection techniques unattainable in a single compartment of the conventional ion cyclotron resonance (ICR) cell. In particular, multi-electrode detection at a multiple of the main cyclotron frequency can be utilized without the loss of sensitivity and other negative effects. The new O-trap designed exclusively for ion detection adds an additional, internal coaxial cylinder around which ions with excited cyclotron orbits rotate. Comparison of simulated performance characteristics of the new O-trap with those of the same-size conventional cylindrical cell shows that the O-trap can provide higher sensitivity and ion capacity. Multiplexing of the O-traps can further increase the analysis speed. Future efforts will be aimed at building and testing experimentally the coaxial O-trap, including optimization of the method of ion transfer between the compartments of the cell.

Computer Simulation↗
PubMed2002

Statistical and deterministic approaches to designing transformations of electrocardiographic leads.

Two different approaches can be used to investigate the relationships among electrocardiographic leads: a statistical one, based on the analysis of recorded electrocardiograms (ECGs), and a deterministic one, based on physical principles that govern the current flow in irregularly shaped volume conductors such as the human body. The purpose of this study was to compare these two approaches. For the statistical investigation, the data set consisted of 120-lead ECGs recorded in a population including normal subjects (n = 290), post-myocardial-infarction patients (n = 497), patients with a history of ventricular tachycardia but no evidence of a previous myocardial infarction (n = 105), and patients with a single-vessel coronary artery disease who underwent coronary angioplasty (n = 91). Lead transformations of interest were obtained by fitting the multiple-regression model to this data set by the least-squares method. For the deterministic investigation, we used a boundary-element model of the human torso to simulate body-surface potentials in response to three orthogonal unit dipoles placed consecutively at 1,239 ventricular source locations, and the resulting body-surface potential distributions (instead of the recorded ECGs) were then fitted by the multiple-regression model. The results suggest that the lead transformations should be preferably designed by statistical analysis of recorded ECGs. Regression models with a small number of predictors (eg, those based on three ECG leads) are the most reliable; those using more predictors are fraught with the danger of collinearity when predictors are highly correlated (as occurs in the standard 12-lead ECG). Model-derived deterministic transformations are compatible with statistically derived ones, provided that the distributed character of the cardiac sources is taken into account. We conclude that statistical associations among electrocardiographic leads can be reliably quantified in sufficiently large and diverse databases of recorded data; the causality of these associations can be supported by appropriate deterministic models based on the laws of physics.

Angioplasty, Balloon, Coronary↗
PubMed1993

In vitro carcinogenesis of mammary epithelial cells by N-nitroso-N-methylurea using a collagen gel matrix culture.

Carcinogenesis is a lengthy process which eventually culminates in the transformed phenotype, cancer. However, much remains to be defined about the process of transformation. In vivo models for the study of the carcinogenic process present limitations because it is not possible to detect the premalignant stages in the animals. An in vitro model, on the other hand, facilitates the study of the carcinogenic process because it enables one to dissect out the crucial events required for carcinogenesis to occur. As carcinogenesis is believed to be a multistep process; initiation, promotion, and progression, a multistep, in vitro system has been devised in our laboratory to mimic each of these stages. We have previously shown the formation of "microtumors" in collagen gels, induced by 7,12-dimethylbenz(a) anthracene. In the present study the direct acting water soluble, mammary carcinogen, N-nitroso-N-methylurea (NMU) was used for tumorigenesis of mammary epithelial cells in culture. Mammary epithelial cells from virgin Sprague-Dawley rats were propagated and exposed to single or multiple doses of NMU while growing as a monolayer in glass petri dishes (initiation). Initiation cells were then plated into a collagen gel matrix culture. Prolonged growth in the collagen gels afforded for the progression of the transformed cells into discernable microtumors in the three-dimensional matrix of the collagen. The morphology of these "tumors" was determined by histologic sections of the gels. Fewer, if any, such structures existed in the untreated gels.

Animals↗
PubMed2006

Expression of renal transforming growth factor-beta and its receptors in a rat model of chronic cyclosporine-induced nephropathy.

OBJECTIVE: We sought to detect expression of transforming growth factor-beta 1 (TGF-beta1) as well as its receptors type I (TRI) and type II (TRII) in rat kidneys during chronic cyclosporine (CsA)-induced nephropathy. METHODS: Twenty four rats were randomly divided into three experimental groups: group 1; NSD (control, n = 8) were administered a normal sodium diet, group 2; LSD (n = 8) were administered a low sodium diet, group 3; CsA (n = 8) were sodium-depleted rats administered Neoral by gastric gavage in a model of chronic CsA-induced nephropathy. TGF-beta1, TRI, and TRII proteins, as well as TRI and TRII mRNAs were measured in the CsA-treated rat kidneys by immunohistochemistry and in situ hybridization, respectively. Semiquantitative results were shown by image analysis. RESULTS: The expression of TGF-beta1, TRI, TRII, TRI mRNA, and TRII mRNA were all increased in CsA-treated rat kidneys, compared with NSD or LSD (P < .05). CONCLUSION: Our study showed that the ligand of TGF-beta1 and its receptors TRI, TRII were all up-regulated. It may be important to inhibit the expression of TGF-beta1 or its receptors in patients who suffer from chronic CsA-induced nephropathy.

Animals↗
PubMed1986

Sequential estimation of genetic and phenotypic parameters in multitrait mixed model analysis.

Single-trait and multitrait (2-, 3-, 4-, and 5-trait) restricted maximum likelihood methods were applied to the same set of data with complete information on all traits. Results suggest that parameter estimates from a data set vary depending upon the type of analysis (single- or multitrait model) and upon the other traits included in multitrait analysis. The choice of parameter estimation method for a breeding design should be based on the breeding goal. In parameter estimation or sire evaluation, traits included in a multitrait analysis should correspond to the traits of interest in the breeding goal. Multitrait analysis explores all intercorrelations simultaneously in parameter estimation and thus provides a complete picture of all interrelationships among traits. In contrast, single-trait analysis produces pairwise (simple) correlations and ignores the possible contribution of other related traits under study to the pairwise correlation. The 5-trait model analysis through canonical transformation was about 300% more efficient in terms of computer time than single-trait model analysis of the same 5 traits. In this study, parameter estimates converged faster under multitrait analysis through canonical transformation than under single-trait analysis.

Animals↗
PubMed2000

Application of a disease-specific, quality-of-life measure (QoL-AGHDA) in growth hormone-deficient adults and a random population sample in Sweden: validation of the measure by rasch analysis.

OBJECTIVE: Growth hormone deficiency (GHD) in adults has been associated with impaired health status and quality of life (QoL) in several studies using generic measures, and in a few studies using recently developed disease-specific measures. Theoretically, disease-specific measures may be more sensitive and succinct than generic measures, and hence prove convenient for general use in clinical practice. The present study sought to validate the scaling properties of the disease-specific QoL-AGHDA measure through the implementation of Rasch analysis. The study also sought to compare, by using the QoL-AGHDA, the QoL of a relatively large Swedish cohort of adults with untreated GHD with that of a reference population also from Sweden. PATIENTS: The QoL of 111 adults with untreated GHD from Stockholm and Göteborg was compared with that of 1448 adult subjects randomly selected from the population of Göteborg. MEASUREMENTS: The scaling properties of the QoL-AGHDA were assessed by investigating its fit to a dichotomous Rasch model. Rasch-transformed QoL scores from the QoL-AGHDA questionnaire were stratified by age and gender, and 95% confidence intervals were calculated. RESULTS: Rasch analysis of the QoL-AGHDA indicated the measure to be robust in terms of its unidimensionality and ordering properties, and lack of differential item functioning. The raw scores produced by the QoL-AGHDA are at the ordinal level. Non-overlapping 95% confidence intervals of Rasch-transformed interval scores in most age categories indicated that men and women with GHD had significantly lower QoL than the reference population. CONCLUSION: The Swedish QoL-AGHDA has good scaling properties, and hence can be considered a robust measure. It is suitable for assessing quality of life in adults with GH deficiency, and for making comparisons with adults who are not growth hormone deficient. Adult GH deficiency is associated with a significant impairment in QoL.

Adult↗
PubMed2001

Reengineering clinical psychiatry in academic medical centers: processes and models of change.

Managed care poses a major threat to the future of clinical psychiatry in academic settings. High costs and an aversion to the business aspects of service delivery have left academic departments at a disadvantage in the marketplace. However, numerous departments have attempted to adapt to the current health care environment. On the basis of a review of these efforts, the authors identify the processes of change and seven models of change being employed to reengineer psychiatry within academic medical centers. The models are labeled transform, build, manage, buy, partner or affiliate, sell or lease, and consult. The authors discuss the strengths and weaknesses of these efforts and suggest three approaches to establishing the unique value of academic clinical services for purchasers and for the public. The first is to enhance productivity and clinical effectiveness through greater integration of clinical care, research, and training. The second is to take a leadership role in managing care, and the third is to advance the integration of behavioral health care with primary medical care and other specialties in the medical center.

Academic Medical Centers↗
PubMed1992

Malignant transformation of human melanocytes: induction of a complete melanoma phenotype and genotype.

Human melanoma provides a model to study malignant transformation and tumor progression. Expression of ras oncogenes in cultured normal human diploid melanocytes has induced a subset of phenotypic traits that are characteristic of malignant melanoma cells, including altered morphology, anchorage independence, induction of class II MHC antigens, up-regulation of the ganglioside GD3, and chromosomal abnormalities. However, other characteristics of melanoma, such as loss of expression of adenosine deaminase-binding protein and tumorigenicity, were not observed. We report here that melanocytes infected with a retrovirus containing the viral Ha-ras oncogene underwent complete transformation, acquiring all phenotypic characteristics of malignant melanomas observed in vivo. Transformation occurred in a sequential manner and was associated with spontaneous chromosomal instability. Cytogenetic analysis of transformed melanocytes indicated that the earliest structural chromosomal abnormalities were isochromosomes 6p and 9q followed by complete loss of chromosome 1p, all common karyotypic abnormalities described in human melanomas. The findings suggest that these chromosome regions which are deleted or relatively deficient may contain genes that are critical for the initiation and progression of the melanoma phenotype.

3T3 Cells↗
PubMed1991

Analysis of poultry fertility data. 2. Comparison of long- and short-term fertility trials.

The purpose of the present study was to determine the effect of egg collection interval on interpretation of fertility trial results. Low-fertility (LF) and randombred (RB) Delaware hens were inseminated with spermatozoa from LF or RB Delaware roosters. Three replicate trials were performed for each cross. Eggs were collected for 21 days following insemination. Fertility was analyzed with a log odds model following logit transformation. Separate analyses were performed on data collected throughout the first 7 days and the entire egg collection interval. Duration of fertility over the 21-day interval was analyzed by iterative least squares. Whereas the 21-day egg collection interval afforded detection of differences between males and between females, no differences were observed following analysis of data from the 7-day interval. Differences in reproductive efficiency also were detected when data were analyzed by iterative least squares. Thus, the value of a fertility trial is, in part, determined by the length of the egg collection interval.

Animals↗
PubMed1987

The heat-stable cytosolic factor that promotes glucocorticoid receptor binding to DNA is neither thioredoxin nor ribonuclease.

Treatment of rat liver cytosol containing temperature-transformed [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). However, transformed receptors that are treated with MMTS and then separated from low Mr components of cytosol by passage through a column of Sephadex G-50 have very little DNA-binding activity when DTT is added to regenerate sulfhydryl moities. The receptors will bind to DNA if whole liver cytosol or boiled liver cytosol is added in addition to DTT. The effect of boiled cytosol is mimicked by purified rat thioredoxin or bovine RNase A in a manner that does not reflect the reducing activity of the former or the catalytic activity of the latter. This suggests that the reported ability of each of these heat-stable peptides to stimulate DNA binding by glucocorticoid receptors is not a biologically relevant action. We suggest that stimulation of DNA binding of partially purified receptors by boiled cytosol does not constitute a reconstitution of a complete cytosolic system in which the dissociated receptor must associate with a specific heat-stable accessory protein required for DNA binding, as has been suggested in the "two-step" model of receptor transformation recently proposed by Schmidt et al. (Schmidt T.J., Miller-Diener, A., Webb M.L. and Litwack G. (1985) J. biol. Chem. 260, 16255-16262).

Animals↗
PubMedDate not supplied

Intervention efficacy and malignant transformation to oral cancer among patients with leukoplakia (Review).

Elimination of leukoplakia is one of the strategies for prevention of oral cancer. However, the efficacy of reducing malignant transformation for the treated leukoplakia has rarely been reported despite a number of studies addressing its malignant transformation to oral cancer after intervention. The obstacle to do so is partially due to different lengths of follow-up and partially due to lack of information pertaining to the disease natural history that can be taken as a standard group for comparison. This work aimed to quantify the progression from leukoplakia to carcinoma with and without intervention after systematic literature review. The overall comparison of the efficacy of intervention across studies was therefore made. The literature between 1934 and 2001 was first reviewed, including two studies addressing the disease natural history model and 15 studies pertaining to intervention model in reducing malignant transformation. The simple constant-incidence exponential model and non-constant incidence models (Weibull and Quadratic models) were therefore applied to estimate annual malignant transformation after intervention in various studies. Annual transition rates pertaining to the disease natural history were also estimated by Markov models. Intervention efficacy index using 10-year cumulative incidence from both models was also developed to assess intervention efficacy across studies. For the disease natural history, the estimates of annual transition rate of leukoplakia, annual transition rate from leukoplakia to oral cancer in the PCDP, and annual transition rate from the PCDP to clinical phase were 0.00121 (0.00019-0.00150), 0.0605 (0.0436-0.0755), and 1.8797 (0.13242-2.4352), respectively. Similar findings were observed in another retrospective study. Annual malignant transformation rates after intervention range from 0.0003 to 0.062 assuming constant incidence. Studies assuming non-constant incidence show different patterns of increasing or decreasing risk with time. The estimates regarding the efficacy of intervention for each study with different follow-up periods ranged from 42.9 to 99.3%. The heterogeneity of intervention efficacy due to a wide range of annual rates of malignant transformation was demonstrated in this review. This implies that prevention program for reducing malignant transformation may vary with different areas. Factors need to be considered including different diagnosis criteria, different histological type, distributions of risk factor, different patient resources, and different compliance rates or insufficient medical intervention.

Carcinoma↗
PubMed2001

Delayed rt-PA treatment in a rat embolic stroke model: diagnosis and prognosis of ischemic injury and hemorrhagic transformation with magnetic resonance imaging.

The authors characterized effects of late recombinant tissue plasminogen activator (rt-PA) administration in a rat embolic stroke model with magnetic resonance imaging (MRI), to assess potential MRI correlates, or predictors, or both, of rt-PA-induced hemorrhage. Diffusion-, perfusion-, and postcontrast T1-weighted MRI were performed between 4 and 9 hours and at 24 hours after embolic stroke in spontaneously hypertensive rats. Treatment with either rt-PA or saline was started 6 hours after stroke. A spectrophotometric hemoglobin assay quantified hemorrhage severity. Before treatment, relative cerebral blood flow index (rCBFi) and apparent diffusion coefficient (ADC) in the ischemic territory were 30% +/- 23% and 60% +/- 5% (of contralateral), respectively, which increased to 45% +/- 39% and 68% +/- 4% 2 hours after rt-PA. After 24 hours, rCBFi and ADC were 27% +/- 27% and 59 +/- 5%. Hemorrhage volume after 24 hours was significantly greater in rt-PA-treated animals than in controls (8.7 +/- 3.7 microL vs. 5.1 +/- 2.4 microL, P < 0.05). Before rt-PA administration, clear postcontrast T1-weighted signal intensity enhancement was evident in areas of subsequent bleeding. These areas had lower rCBFi levels than regions without hemorrhage (23% +/- 22% vs. 36% +/- 29%, P < 0.05). In conclusion, late thrombolytic therapy does not necessarily lead to successful reperfusion. Hemorrhage emerged in areas with relatively low perfusion levels and early blood-brain barrier damage. Magnetic resonance imaging may be useful for quantifying effects of thrombolytic therapy and predicting risks of hemorrhagic transformation.

Animals↗
PubMed2005

v-Jun downregulates the alpha 2 (I) collagen target gene indirectly through Sp1/3.

Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position -1080 to +109, was performed. Transient transfections with -1080/+109 and deleted derivatives revealed that a short proximal fragment, -433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of -433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun-Sp1/3-DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3.

Animals↗
PubMed1984

Expression of cellular oncogenes in human malignancies.

Cellular oncogenes have been implicated in the induction of malignant transformation in some model systems in vitro and may be related to malignancies in vivo in some vertebrate species. This article describes a study of the expression of 15 cellular oncogenes in fresh human tumors from 54 patients, representing 20 different tumor types. More than one cellular oncogene was transcriptionally active in all of the tumors examined. In 14 patients it was possible to study normal and malignant tissue from the same organ. In many of these patients, the transcriptional activity of certain oncogenes was greater in the malignant than the normal tissue. The cellular fes (feline sarcoma) oncogene, not previously known to be transcribed in mammalian tissue, was found to be active in lung and hematopoietic malignancies.

Adenocarcinoma↗
PubMed1992

Simian virus 40 (SV40) large T antigen-dependent amplification of an Epstein-Barr virus-SV40 hybrid shuttle vector integrated into the human HeLa cell genome.

We analysed the DNA rearrangements that occurred during the integration and amplification of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector in human cells. The human HeLa cell line was episomally transformed with the EBV-SV40 p205-GTI plasmid. After a 2 month culture in a selective medium, a HeLa cell-derived population (H-G1 cells) was obtained in which the p205-GTI vector was integrated as a single intact copy deleted in the EBV latent origin of replication (OriP). Sequencing data showed that the endpoints of the plasmid sequences, at the plasmid-cell DNA junctions, are located within the two essential elements of EBV OriP, which may form several secondary structures. This result suggests that a specific DNA sequence (OriP) or palindromic structures could play a role in this integration process. This represents the first fully characterized site of integration of an EBV vector in human cells. The transient expression of the SV40 large T antigen in H-G1 cells leads to the appearance of episomal molecules with an extremely heterogeneous size pattern. Individual analysis of these episomes after rescue in bacteria indicated that they retained sequences of both the p205-GTI plasmid and cellular DNA. Comparison of the structure of these circular DNAs with those of the integrated p205-GTI copy indicated that large T antigen expression in human cells leads to the amplification of the integrated shuttle vector according to the 'onion skin' model developed for transformed rodent cells. Indeed, amplified sequences were colinear with the integrated p205-GTI copy and its surrounding cellular sequences, distributed almost equally around the SV40 replication origin, and circularized by illegitimate recombination which did not involve specific nucleotide sequences. This system is of interest in that it enables easy recovery of individual recombined molecules in host bacteria. Each isolated clone contains a unique recombination junction which is easily and rapidly characterized and sequenced.

Antigens, Polyomavirus Transforming↗
PubMed1983

Transformation and immortalization of human keratinocytes by SV40.

We have studied the appearance of transformed properties following infection of human epidermal keratinocytes by the oncogenic virus SV40. Shortly after infection, only a small fraction of the cells are positive for SV40 T antigen by immunofluorescence; this fraction progressively increases upon serial subcultivation concomitant with an increase in plating/colony-forming efficiency and growth rate. The capacity of the cells to differentiate progressively decreases, as indicated by cytochemical staining and cornified cell-envelope formation induced by suspension in methyl cellulose. The infected cells enter a period of growth crisis characterized by cytopathology and cell death as the level of T antigen synthesis reaches about 90 percent positive cells at about the tenth serial passage. Viable cells emerging from the crisis period are found to exhibit anchorage-independent growth, as indicated by the formation of viable colonies in semisolid media, but there is considerable variability in colony formation among clones isolated from anchorage-independent populations. The emergent population also manifests phenotypic instability in terms of the appearance of variants, which, in contrast to uninfected cells, expresses a well-defined actin cytoskeleton. The infected cells eventually become "immortalized," as evidenced by an indefinite lifespan, i.e., replication capability maintained well beyond the ordinary time of senescence for uninfected cells. We present these findings in the context of a stage-specific model of epithelial transformation in vitro.

Actins↗
PubMed1994

Growth suppression of normal mammary epithelial cells by wild-type p53.

p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.

Alleles↗
PubMed1974

Kinetic constants determined from membrane transport measurements: carbonic anhydrase activity at high concentrations.

Facilitated diffusion rates can be used to determine kinetic constants for rapid reactions occurring within membranes and thin fluid layers. We have applied this technique to the study of the reversible CO(2) hydration reactions catalyzed by carbonic anhydrase (EC 4.2.1.1; carbonate hydro-lyase). The experimental method entails the diffusion of tracer (14)CO(2) through Millipore filter membranes impregnated with aqueous bicarbonate solutions containing various concentrations of dissolved enzyme. A mathematical model of the simultaneous diffusion/reaction transport process is analyzed to predict the effective diffusion rate in terms of the relevant kinetic parameters. The solution to the mathematical model can be transformed to yield straight-line relations analogous to Lineweaver-Burk plots. The pseudo-first-order enzymatic rate constant for the hydration reaction can be determined from the slope or intercept of a plot of this straight-line relationship. Rate constants were accurately measured at high enzyme concentrations for reactions having half-times under a millisecond. The rate constants agree well with other reported kinetic constants for carbonic anhydrase, and the known pH-activity dependence and bicarbonate inhibition are quantitatively demonstrated. The specific activity is constant up to 4.0 mg/ml, which is believed to be the highest concentration at which the activity has been measured. The membrane transport technique has general applicability for other rapid reaction systems.

Bicarbonates↗