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PubMed1999

Why and how is soft copy reading possible in clinical practice?

The properties of the human visual system (HVS) relevant to the diagnostic process are described after a brief introduction on the general problems and advantages of using soft copy for primary radiology interpretations. At various spatial and temporal frequencies the contrast sensitivity defines the spatial resolution of the eye-brain system and the sensitivity to flicker. The adaptation to the displayed radiological scene and the ambient illumination determine the dynamic range for the operation of the HVS. Although image display devices are determined mainly by state-of-the-art technology, analysis of the HVS may suggest technical characteristics for electronic displays that will help to optimize the display to the operation of the HVS. These include display size, spatial resolution, contrast resolution, luminance range, and noise, from which further consequences for the technical components of a monitor follow. It is emphasized that routine monitor quality control must be available in clinical practice. These image quality measures must be simple enough to be applied as part of the daily routine. These test instructions might also serve as elements of technical acceptance and constancy tests.

Adaptation, Ocular↗
PubMed1999

Chemical or biological activity in open chaotic flows.

We investigate the evolution of particle ensembles in open chaotic hydrodynamical flows. Active processes of the type A+B-->2B and A+B-->2C are considered in the limit of weak diffusion. As an illustrative advection dynamics we consider a model of the von Kármán vortex street, a time-periodic two-dimensional flow of a viscous fluid around a cylinder. We show that a fractal unstable manifold acts as a catalyst for the process, and the products cover fattened-up copies of this manifold. This may account for the observed filamental intensification of activity in environmental flows. The reaction equations valid in the wake are derived either in the form of dissipative maps or differential equations depending on the regime under consideration. They contain terms that are not present in the traditional reaction equations of the same active process: the decay of the products is slower while the productivity is much faster than in homogeneous flows. Both effects appear as a consequence of underlying fractal structures. In the long time limit, the system locks itself in a dynamic equilibrium state synchronized to the flow for both types of reactions. For particles of finite size an emptying transition might also occur leading to no products left in the wake.

Journal Article↗
PubMed1995

Interaction of integrin alpha IIb beta 3 with multiple fibrinogen domains during platelet adhesion.

We have investigated how modulation of integrin alpha IIb beta 3 function influences the mechanisms that initiate platelet thrombus formation onto surface-bound fibrinogen and isolated fibrinogen domains. Under stationary conditions and with full activation of platelets blocked by prostaglandin E1, the carboxyl-terminal gamma 400-411 sequence is necessary for establishing initial contact with the immobilized substrate. Molecules containing a single copy of this sequence, like the plasmin-generated fibrinogen fragment D, support platelet spreading, but the resulting attachment to the surface is loose and disrupted by minimal peeling force. In contrast, platelets adhere firmly to intact fibrinogen under the same conditions, suggesting that recognition of contact sites outside a single D domain can secure the firm interaction not supported by a single gamma 400-411 sequence. If platelets are activated, the gamma 400-411 sequence is no longer necessary to initiate the adhesion process but becomes sufficient, even as a single copy, to mediate stable surface attachment in the absence of shear stress. Under conditions of flow, however, intact fibrinogen but not fragment D can support adhesion, regardless of whether platelets have the potential to become activated or not. These results indicate the functional relevance of multiple fibrinogen domains during the initial stages of the platelet adhesion process.

Alprostadil↗
PubMed1987

A microcomputer-based system for processing 31-phosphorus nuclear magnetic resonance spectra from studies of cardiac metabolism in immature hearts.

We designed an interactive microcomputer-based digital data processing system for analysis of 31-phosphorus nuclear magnetic resonance (31P-NMR) spectra from studies of cardiac metabolism in immature and neonatal hearts. This system included a digitizing tablet (Kurta Series Two), a microcomputer (IBM PC XT) and a graphics plotter (Hewlett-Packard 7470A) used in conjunction with a Nicolet 1280 NMR signal processing computer. We obtained 31P spectra from isolated perfused rabbit hearts with a Nicolet NT-200 4.7 Tesla superconducting NMR spectrometer operated in the pulsed Fourier transform mode. The small size of the hearts resulted in increased noise in spectra and demanded comparison of methods used to quantitate changes in inorganic phosphorus, phosphocreatine and ATP during ischemic stress. We performed microcomputer operations and interfacing functions with a software package written in BASIC. This system simplified documentation, data filing and statistical data processing. Our microcomputer system displayed and made hard copies of digitized spectra and results of analyses. Errors in data entry were rectified directly with this program. Consistent data reduction improved the precision of the physiological results and reduced the influence of noise on 31P spectra from neonatal hearts weighing about 0.5 g. The system flexibility extends its application to NMR spectra analysis for other in vivo organ systems, and signal processing in other biological research.

Animals↗
PubMed1994

Interhemispheric cooperation during lexical processing is mediated by the corpus callosum: evidence from the split-brain.

If two copies of a meaningful word are tachistoscopically presented simultaneously in both visual half-fields of normal subjects the word will be processed more rapidly and more accurately compared to unilateral presentation (bilateral gain). The word-specific bilateral gain may be due to excitatory transcallosal connections within interhemispheric cell assemblies corresponding to words. In this case, the bilateral gain should be absent in split-brain patients. L.B., a split-brain patient, performed a lexical decision task with words and non-words presented in the left visual field, the right visual field, or in both visual fields simultaneously. As predicted, bilateral presentations did not improve performance compared to unilateral presentation in the right visual field. This result suggests that transcallosal connections play a significant role in lexical processing.

Adult↗
PubMed2006

Fast protein evolution and germ line expression of a Drosophila parental gene and its young retroposed paralog.

This is the first detailed study of the evolution, phylogenetic distribution, and transcription of one young retroposed gene, CG13732, and its parental gene CG15645, whose functions are unknown. CG13732 is a recognizable retroposed copy of CG15645 retaining the signals of this process. We name the parental gene Cervantes and the retrogene Quijote. To determine when this duplication occurred and the phylogenetic distribution of Quijote, we employed polymerase chain reaction, Southern blotting, and the available information on sequenced Drosophila genomes. Interestingly, these analyses revealed that Quijote is present only in 4 species of Drosophila (Drosophila melanogaster, Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana) and that retroposed copies of Cervantes have also originated in the lineages leading to Drosophila yakuba and Drosophila erecta independently in the 3 instances. We name the new retrogene in the D. yakuba lineage Rocinante and the new retrogene in the D. erecta lineage Sancho. In this work, we present data on Quijote and its parental gene Cervantes. Polymorphism analysis of the derived gene and divergence data for both parental and derived genes were used to determine that both genes likely produce functional proteins and that they are changing at a fast rate (KA/KS approximately 0.38). The negative value of H of Fay and Wu in the non-African sample reveals an excess of derived variants at high frequency. This could be explained either by positive selection in the region or by demographic effects. The comparative expression pattern shows that both genes express in the same adult tissues (male and female germ line) in D. melanogaster. Quijote is also expressed in male and female in D. simulans, D. sechellia, and D. mauritiana. We argue that the fast rate of evolution of these genes could be related to their putative germ line function and are further studying the independent recruitment of Cervantes-derived retrogenes in multiple lineages.

Animals↗
PubMed1994

Suppression of yeast RNA polymerase III mutations by FHL1, a gene coding for a fork head protein involved in rRNA processing.

The FHL1 gene was isolated by screening for high-copy-number suppressors of conditional RNA polymerase III mutations. This gene is unique on the yeast genome and was located close to RPC40 and PRE2 on the right arm of chromosome XVI. It codes for a 936-amino-acid protein containing a domain similar to the fork head DNA-binding domain, initially found in the developmental fork head protein of Drosophila melanogaster and in the HNF-3 family of hepatocyte mammalian transcription factors. Null mutations caused a severe reduction in growth rate and a lower rRNA content that resulted from defective rRNA processing. There was no detectable effect on mRNA splicing. Thus, the Fhl1p protein plays a key role in the control of rRNA processing, presumably by acting as a transcriptional regulator of genes specifically involved in that process. Moreover, mutants carrying the RNA polymerase III mutations were slightly defective in rRNA processing. This accounts for the isolation of FHL1 as a dosage-dependent suppressor and suggests that rRNA processing depends on a still-unidentified RNA polymerase III transcript.

Amino Acid Sequence↗
PubMed2005

Cross-typic specificity and immunotherapeutic potential of a human HPV16 E7-specific CTL line.

Cervical cancer (CaCx) is strongly associated with human papillomavirus (HPV) infection, particularly HPV types 16 and 18. The constitutive expression of HPV E6 and E7 proteins in CaCx makes them attractive targets for CTL based immunotherapy. However cervical carcinomas may have features, e.g., antigen processing defects, that limit the effectiveness of HPV specific CTL. Furthermore most vaccine development has concentrated on HPV type 16, and it is not clear whether such vaccines could induce CTL able to cross-react on related oncogenic HPV types, e.g., HPV31 and 52. To investigate these potentially important parameters in vitro, we used a CTL (D4) specific for HPV16 E7(11-20). D4 was able to kill a variety of HPV16+ CaCx cell lines including those with suspected (CaSki) or known antigen processing defects (C33A), and with low HPV DNA copy number (SiHa). D4 was also able to cross react on a related peptide from HPV52 E7 but not HPV31 E7. Further analysis suggested that D4 cross reactivity against related peptides was influenced both by TCR contact residues and a certain threshold for peptide binding. The HPV cross-reactivity was confirmed at the whole protein level as D4 was also able to recognize the endogenously processed forms of HPV16 and 52 E7 but not 31 E7. These results suggest that HPV16 E7(11-20) would be a useful epitope for immunotherapy in both HPV 16 and 52 tumours. Despite this, it is difficult to generate these CTL in response to vaccination, emphasizing the need for definition of novel epitopes and more efficient vaccination strategies.

Cell Line, Tumor↗
PubMed1996

Differential coexpression of genes encoding prothyrotropin-releasing hormone (pro-TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: implications for differential processing of pro-TRH.

Pro-TRH is cleaved at paired basic residues to yield five copies of TRH and cryptic peptides. Recent studies have shown that the prohormone convertases, PC1 and PC2, can process pro-TRH correctly. To determine whether these two enzymes could play a role in pro-TRH processing in vivo, the regional and cellular colocalization of pro-TRH messenger RNA (mRNA) with the mRNAs encoding the prohormone convertases PC1 and PC2 was examined in rat brain, using in situ hybridization histochemistry. Differential regional distribution of pro-TRH mRNA with PC1 and/or PC2 mRNA was found in several brain regions. For example, in the olfactory regions, there was coexpression of pro-TRH mRNA in the glomerular layer with PC2 mRNA, but not PC1 mRNA, whereas in the tenia tecta, coexpression of pro-TRH and PC1 mRNAs was evident, but PC2 mRNA was absent. Pro-TRH mRNA in the paraventricular nucleus was coexpressed with both PC1 and PC2 mRNAs, whereas the basal lateral hypothalamus showed coexistence of pro-TRH mRNA with PC2 mRNA, but not PC1 mRNA. Interestingly, pro-TRH was expressed in the thalamic reticular nucleus, but neither PC1 nor PC2 was detectable in this region. Cellular colocalization studies using double in situ hybridization histochemistry showed the presence of PC2 mRNA in the pro-TRH neurons of the olfactory glomerular layer and basal lateral hypothalamus, and PC1 mRNA in the pro-TRH neurons in the paraventricular nucleus. These results suggest that PC1 and PC2 are enzyme candidates for the processing of pro-TRH in vivo. Moreover, the differential distribution of PC1 and PC2 mRNAs with pro-TRH mRNA may be responsible for the differential processing of this prohormone in the central nervous system. The absence of PC1 and PC2 mRNAs in certain TRH neurons raises the possibility that prohormone convertases other than PC1 and PC2 may be involved in the processing of brain pro-TRH.

Animals↗
PubMed2004

Kinetics of plasmid segregation in Escherichia coli.

Low copy-number bacterial replicons occupy specific locations in their host cells. Production of a GFP-Lac repressor hybrid protein in cells carrying F or P1 plasmids tagged with a lac operator array reveals that in smaller (younger) cells these plasmids are seen mainly as a single fluorescent focus at mid-cell, whereas larger cells tend to have two foci, one at each quarter-cell position. Duplication of the central focus is presumed to represent active partition of plasmid copies. We report here our investigation by time-lapse microscopy of the subsequent movement of these copies to the quarter positions. Following duplication of the central focus, the new foci migrated rapidly and directly to their quarter-cell destinations, where they remained until the next cell cycle. The speed of movement was about five times faster than poleward migration of oriC and 50 times faster than cell elongation. Aberrant positioning of mini-F lacking its sopC centromere demonstrated the requirement for the partition system in this localization process. From the measured number of F plasmid copies per cell it appears that each migrating focus contains two or more plasmid molecules. The molecular basis of this clustering, and evidence for phasing of the partition event in the cell cycle, are discussed.

Animals↗
PubMed2005

Deubiquitination by proteasome is coordinated with substrate translocation for proteolysis in vivo.

The 26S proteasome mediates degradation of protein substrates labeled with polyUb chains. After recognition by the 19S proteasome regulatory complex, polyUb chains are disassembled and substrates are processed in the 20S core of proteasome. However, the exact relationship of degradation-associated deubiquitination to substrate processing remains unclear. Here, using Ub-based tagging strategies, we provided evidence that removable polyUb chains serve as the signal for proteolytic processing of ubiquitinated substrates. We showed that inhibition of the proteasome by proteasome inhibitor MG132 results in trapping of the substrate in the proteasome. Such a trapping allows proteasomal cleavage of attached non-removable Ub mutant (UbV75,76), which is otherwise a "difficult" deubiquitination substrate. Characterization of deubiquitination and degradation intermediates, generated due to incomplete proteolytic inhibition, revealed changes in proteolytic cleavage sites, within the Gal4-VP16 model substrate, suggesting that the copy number of attached UbV75,76 affects substrate processing. Conversion of lysine48 to arginine48 in UbV75,76 did not have significant effect on in vivo polyubiquitination of multiple Ub-fused substrates, but considerably reduced proteolytic intermediates. Taken together, the results support a model in which deubiquitination process is a crucial event for proteolysis of ubiquitinated substrates and such an event is coordinated with substrate translocation.

Amino Acid Substitution↗
PubMed1990

[Extrachromosomal DNA in mammalian cells].

Extrachromosomal DNA (eDNA) is mammalian cells is represented not only as mitochondrial DNA but also as circular DNA existing both in the nucleus and in the cytoplasm. According to its size eDNA could be divided into two major classes: small polydisperse circular DNAs and large circular DNAs. Small polydisperse circular DNAs sized from 150 to 20,000 base pair are found both in the nucleus and in the cytoplasm. Large circular DNAs from 150,000 to 900,000 base pair are found in the nucleus only. Small polydisperse circular DNAs contain mainly repetitive sequences but occasionally they may contain very low repetitive sequences or unique ones. Such sequences may result from intrachromosomal recombinations, replicon misfiring, reverse transcription or fragmentation of chromosomal DNA by endogenous nucleases. Large circular DNAs are known to consist mainly of amplified sequences of oncogenes or MDR genes, which result from the deletion of appropriate regions of chromosomes. The number of copies of eDNA is greater in the malignant cells and in cells at certain stages of differentiation. The increase in copy number of eDNA reflects the intensity of processes in the cells. Autonomous replication of small and large circular DNAs is evident in tumor cells, although not all eDNAs can replicate autonomously. The presence in tumor cells of self-replicating molecules of eDNA, which are distributed in daughter cells independently of chromosomal DNA, determines presumably the high rate of adaptability of these cells to environmental conditions (metastasis and resistance to chemotherapeutic agents). It is quite possible that the existence of amplified copies of oncogenes and some other genes in the form of eDNA can determine some phenotypic characteristics of the transformed cells.

Animals↗
PubMed2000

DNA segregation in bacteria.

Segregation of DNA in bacterial cells is an efficient process that assures that every daughter cell receives a copy of genomic and plasmid DNA. In this review, we focus primarily on observations in recent years, including the visualization of DNA and proteins at the subcellular level, that have begun to define the events that separate DNA molecules. Unlike the process of chromosome segregation in higher cells, segregation of the bacterial chromosome is a continuous process in which chromosomes are separated as they are replicated. Essential to separation is the initial movement of sister origins to opposite ends of the cell. Subsequent replication and controlled condensation of DNA are the driving forces that move sister chromosomes toward their respective origins, which establishes the polarity required for segregation. Final steps in the resolution and separation of sister chromosomes occur at the replication terminus, which is localized at the cell center. In contrast to the chromosome, segregation of low-copy plasmids, such as Escherichia coli F, P1, and R1, is by mechanisms that resemble those used in eukaryotic cells. Each plasmid has a centromere-like site to which plasmid-specified partition proteins bind to promote segregation. Replication of plasmid DNA, which occurs at the cell center, is followed by rapid partition protein-mediated separation of sister plasmids, which become localized at distinct sites on either side of the division plane. The fundamental similarity between chromosome and plasmid segregation-placement of DNA to specific cell sites-implies an underlying cellular architecture to which both DNA and proteins refer.

Cell Compartmentation↗
PubMed2003

Regulation of the sequential processing of Semliki Forest virus replicase polyprotein.

The replication of most positive-strand RNA viruses and retroviruses is regulated by proteolytic processing. Alphavirus replicase proteins are synthesized as a polyprotein, called P1234, which is cleaved into nsP1, nsP2, nsP3, and nsP4 by the carboxyl-terminal protease domain of nsP2. The cleavage intermediate P123+nsP4 synthesizes minus-strand copies of the viral RNA genome, whereas the completely processed complex is required for plus-strand synthesis. To understand the mechanisms responsible for this sequential proteolysis, we analyzed in vitro translated Semliki Forest virus polyproteins containing noncleavable processing sites or various deletions. Processing of each of the three sites in vitro required a different type of activity. Site 3/4 was cleaved in trans by nsP2, its carboxyl-terminal fragment Pro39, and by all polyprotein proteases. Site 1/2 was cleaved in cis with a half-life of about 20-30 min. Site 2/3 was cleaved rapidly in trans but only after release of nsP1 from the polyprotein exposing an "activator" sequence present in the amino terminus of nsP2. Deletion of amino-terminal amino acids of nsP2 or addition of extra amino acid residues to its amino terminus specifically inhibited the protease activity that processes the 2/3 site. This sequence of delayed processing of P1234 would explain the accumulation of P123 plus nsP4, the early short-lived minus-strand replicase. The polyprotein stage would allow correct assembly and membrane association of the RNA-polymerase complex. Late in infection free nsP2 would cleave at site 2/3 yielding P12 and P34, the products of which, nsP1-4, are distributed to the plasma membrane, nucleus, cytoplasmic aggregates, and proteasomes, respectively.

Cysteine Endopeptidases↗
PubMed2005

A rapid real-time qRT-PCR assay for ovine beta-actin mRNA.

Beta-Actin mRNA is often used for normalization in gene expression experiments. We describe a sensitive, rapid and specific quantitative assay for the cytoplasmic ovine beta-actin mRNA. The assay was based on the polymerase chain reaction (PCR) with real-time fluorescence resonance energy transfer (FRET) measurements to amplify cDNA products reverse transcribed from mRNA. A part of the ovine beta-actin sequence was amplified from cDNA from fetal ovine synovial (FOS) cells with mRNA-specific primers and cloned into a plasmid clone. The assay standard curve was constructed with dilutions of this plasmid. The assay was linear over five orders of magnitude and detected down to 600 copies per reaction of target DNA. Intraassay coefficient of variation was 12%. Detection of the beta-actin gene was eliminated by designing FRET probes at splice junctions and detection of putative processed pseudogenes was minimized by using FRET assay design with four oligonucleotides. We measured 0.2 copies per cell in RNA preparations without reverse transcription and DNase digestion. This might represent processed pseudogenes. In constrast, we measured 1400 beta-actin mRNA copies per cell in RNA preparations after the RT and DNase steps. The assay should, therefore, be sensitive enough to measure beta-actin from a single individual cell. Dilution of target DNA in murine RNA or ovine cDNA preparations did not effect efficiency of PCR or linearity of the assay. The quantitative assay described in this work can be used to correct for variations in various real-time qRT-PCR experiments in ovine cells with diverse goals, including gene expression studies, quantitation of viral load in infected cells and in various gene therapy experiments measuring vector load and expression in transduced cells.

Actins↗
PubMed1995

[Digitized medical images].

Digitalization of medical images implies that we are able to process images by computers, are able to make as many as exact copies of original images, and are able to transfer images without any loss of information. Clinical usefulness of image processing with special attention to three dimentional images were discussed. There are many problem to be solved in the development of PACS. It is important at least now that discussion of images for primary diagnosis and images for reference should be done separately. Utilization of digitized images for education is promising. A trial of education using a world wide web server-client system was reported. Possible trends of digitized medical images were described.

Diagnosis, Computer-Assisted↗
PubMed2004

The role of perceptual bias in complex figure recall.

Several studies have demonstrated that constructional and organizational abilities affect how well visual stimuli are remembered. The purpose of the present study was to determine whether perceptual bias, independent of constructional ability, influenced visual memory performance. Participants were 118 normal adults who were administered the Rey-Osterrieth Complex Figure (ROCF) and a similarity judgement task requiring then to decide which of two hierarchically constructed comparison figures most resemble a standard figure. The initial steps of a multiple regression analysis indicated that delayed recall of the ROCF was directly related to copy accuracy, inversely related to age, and better in males than females. In the final step of the regression model, performance on the similarity judgement task also predicted ROCF recall, with a more global perceptual bias predicting better recall. Results indicate that even when copy accuracy and demographic variables are accounted for, components of visual perception influence how well visual stimuli are encoded and recalled. Findings are consistent with research correlating global processing and spatial ability, and suggest that in addition to copy ability and organization, visuoperceptual bias must be considered as a moderator variable when interpreting performance as a visual memory test.

Adult↗
PubMed2004

Social learning strategies.

In most studies of social learning in animals, no attempt has been made to examine the nature of the strategy adopted by animals when they copy others. Researchers have expended considerable effort in exploring the psychological processes that underlie social learning and amassed extensive data banks recording purported social learning in the field, but the contexts under which animals copy others remain unexplored. Yet, theoretical models used to investigate the adaptive advantages of social learning lead to the conclusion that social learning cannot be indiscriminate and that individuals should adopt strategies that dictate the circumstances under which they copy others and from whom they learn. In this article, I discuss a number of possible strategies that are predicted by theoretical analyses, including copy when uncertain, copy the majority, and copy if better, and consider the empirical evidence in support of each, drawing from both the animal and human social learning literature. Reliance on social learning strategies may be organized hierarchically, their being employed by animals when unlearned and asocially learned strategies prove ineffective but before animals take recourse in innovation.

Adaptation, Psychological↗