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PubMed2001

Conditioning in the rat: an in vivo model to investigate the molecular mechanisms and clinical implications of brain-immune communication.

A wealth of in vitro and ex vivo evidence has described both close anatomical interaction and functional bi-directional communication between the immune and central nervous systems (CNS). These data have provided a framework for understanding the physiological mechanisms whereby behavioural factors may impact immune-related disease. An understanding of this interaction, however, as well as verification of the biological relevance of communication among these systems, requires in vivo animal modelling. The development of psychoneuroimmunological models in the laboratory rat has played a key role in advancing the understanding of the influence of behaviour on immune status. One such paradigm is the behavioural conditioning of immune function in the rat. This elegant model is characterised by the ability to examine simultaneously both afferent and efferent brain-immune communication. Specifically, the role of peripheral cytokines in signalling the brain, as well as their anatomical and cellular targets in the CNS, can be identified. On the other hand, the neural and humoral pathways whereby the CNS influences the function and distribution of peripheral immunocytes can be demonstrated, together with the target hormone receptors on immunocompetent cells. Finally, the in vivo biological relevance of brain-immune communication is revealed by behavioural conditioning, demonstrating that clinically relevant conditions such as heart allograft survival can be modified by behavioural processes. Behavioural conditioning thereby provides an excellent example of the utility of in vivo laboratory rat models in psychoneuroimmunology research. Such paradigms not only provide a more complete knowledge of CNS-immune system interaction, but are the platform for determining potential clinical application of this information.

Animals↗
PubMed2000

Confirmation of a unique intra-dimer cooperativity in the human hemoglobin alpha(1)beta(1)half-oxygenated intermediate supports the symmetry rule model of allosteric regulation.

The contribution of the alpha(1)beta(1)half-oxygenated tetramer [alphabeta:alphaO(2)betaO(2)] (species 21) to human hemoglobin cooperativity was evaluated using cryogenic isoelectric focusing. The cooperative free energy of binding, reflecting O(2)-driven protein structure changes, was measured as (21)DeltaG(c) = 5.1 +/- 0. 3 kcal for the Zn/FeO(2) analog. For the Fe/FeCN analog, (21)DeltaG(c) was estimated as 4.0 kcal after correction for a CN ligand rearrangement artifact, demonstrating that ligand rearrangement does not invalidate previous conclusions regarding this species. In the context of the entire Hb cooperativity cascade, which includes eight intermediate species, the 21 tetramer is highly abundant relative to the other doubly-ligated species, providing strong support for the previously determined consensus partition function of O(2) binding and for the Symmetry Rule model of hemoglobin cooperativity (Ackers et al., Science 1992;255:54-63). Cooperativity of normal human hemoglobin is shown to depend on site-configuration, and not solely the number of O(2) bound, nor the occupancy of alpha vs. beta subunits. Verification of a unique contribution from the alpha(1)beta(1)doubly-oxygenated species to the equilibrium O(2) binding curve strongly reinforces the Symmetry Rule interpretation that the alpha(1)beta(1)dimer acts both as a structural and functional element in cooperative O(2) binding.

Allosteric Regulation↗
PubMed2006

[Film dose analysis system and its application in radiotherapy verification].

OBJECTIVE: To propose a new computer software-based medical image processing technique with high resolution digital scanner for radiotherapy verification. METHODS: Under the platform of Windows 2000, the software, programmed with Visual C++.NET, was developed according to modular design. All data of the films and the treatment planning system could be processed as images or dose curves for a robust result. RESULTS: Many functions such as data acquisition, automatic analysis and computation and image processing were integrated in the system. Both geometric and dosimetric errors could be calculated at the same time to verify the radiotherapy accuracy. CONCLUSION: This system has good accuracy and stability for cost-effective radiotherapy verification.

Film Dosimetry↗
PubMed2003

Cytokeratin 20 and guanylyl cyclase C mRNA is largely present in lymph node and liver specimens of colorectal cancer patients.

The aim of our prospective study was to detect circulating epithelial cells (CEC) indicating the presence of disseminated tumor cells (DTC) in tissues affected by lymphatic and hematogenic colorectal cancer metastasis. DTC were tracked in lymph node, liver or bone marrow samples of 245 colorectal cancer patients using 2 independent RT-PCR assays for cytokeratin 20 (CK20) and guanylylcyclase C (GCC) that demonstrated a sensitivity of 1 colorectal cancer cell in 10(6) nucleated hematopoietic cells. CK20 mRNA was detected in 79% of lymph nodes, 35% of both liver lobes and 11% of bone marrow samples. GCC mRNA was found in 68% of lymph nodes, 60% of both liver lobes and 6% of bone marrow specimens. Both markers were recorded in 63% of lymph nodes, 45% of at least 1 liver lobe and 1% of bone marrow samples. There was no significant difference when comparing lymph node samples tested positive for both markers in patients with (N1/2; 65%) and without (N0; 56%) nodal involvement. The same was true when comparing the percentages of patients with and without clinically overt distant metastasis who were positive for both markers in at least 1 liver lobe (62% vs. 41%) or in bone marrow (4% vs. 0%). A score denoting the cumulative sum of tests indicating presence of CK20 and GCC mRNA in the liver was significantly related with UICC classification (p = 0.039). However, addition of lymph node results to this score decreased the correlation. The high incidence of clinically inconspicuous lymph node and liver samples tested positive for both markers emphasizes the function of these organs as primary filters for epithelial cells possibly shed from colorectal carcinomas. The potential prognostic significance of these findings warrants verification, especially regarding the importance of CEC or DTC resident in the liver of colorectal cancer patients.

Aged↗
PubMed1996

New approaches in molecular structure prediction.

In the past years, much effort has been put on the development of new methodologies and algorithms for the prediction of protein secondary and tertiary structures from (sequence) data; this is reviewed in detail. New approaches for these predictions such as neural network methods, genetic algorithms, machine learning, and graph theoretical methods are discussed. Secondary structure prediction algorithms were improved mostly by considering families of related proteins; however, for the reliable tertiary structure modeling of proteins, knowledge-based techniques are still preferred. Methods and examples with more or less successful results are described. Also, programs and parameterizations for energy minimisations, molecular dynamics, and electrostatic interactions have been improved, especially with respect to their former limits of applicability. Other topics discussed in this review include the use of traditional and on-line databases, the docking problem and surface properties of biomolecules, packing of protein cores, de novo design and protein engineering, prediction of membrane protein structures, the verification and reliability of model structures, and progress made with currently available software and computer hardware. In summary, the prediction of the structure, function, and other properties of a protein is still possible only within limits, but these limits continue to be moved.

Chemical Phenomena↗
PubMed1999

cDNA microarrays detect activation of a myogenic transcription program by the PAX3-FKHR fusion oncogene.

Alveolar rhabdomyosarcoma is an aggressive pediatric cancer of striated muscle characterized in 60% of cases by a t(2;13)(q35;q14). This results in the fusion of PAX3, a developmental transcription factor required for limb myogenesis, with FKHR, a member of the forkhead family of transcription factors. The resultant PAX3-FKHR gene possesses transforming properties; however, the effects of this chimeric oncogene on gene expression are largely unknown. To investigate the actions of these transcription factors, both Pax3 and PAX3-FKHR were introduced into NIH 3T3 cells, and the resultant gene expression changes were analyzed with a murine cDNA microarray containing 2,225 elements. We found that PAX3-FKHR but not PAX3 activated a myogenic transcription program including the induction of transcription factors MyoD, Myogenin, Six1, and Slug as well as a battery of genes involved in several aspects of muscle function. Notable among this group were the growth factor gene Igf2 and its binding protein Igfbp5. Relevance of this model was suggested by verification that three of these genes (IGFBP5, HSIX1, and Slug) were also expressed in alveolar rhabdomyosarcoma cell lines. This study utilizes cDNA microarrays to elucidate the pattern of gene expression induced by an oncogenic transcription factor and demonstrates the profound myogenic properties of PAX3-FKHR in NIH 3T3 cells.

3T3 Cells↗
PubMed2004

Identification and functional analysis of 'hypothetical' genes expressed in Haemophilus influenzae.

The progress in genome sequencing has led to a rapid accumulation in GenBank submissions of uncharacterized 'hypothetical' genes. These genes, which have not been experimentally characterized and whose functions cannot be deduced from simple sequence comparisons alone, now comprise a significant fraction of the public databases. Expression analyses of Haemophilus influenzae cells using a combination of transcriptomic and proteomic approaches resulted in confident identification of 54 'hypothetical' genes that were expressed in cells under normal growth conditions. In an attempt to understand the functions of these proteins, we used a variety of publicly available analysis tools. Close homologs in other species were detected for each of the 54 'hypothetical' genes. For 16 of them, exact functional assignments could be found in one or more public databases. Additionally, we were able to suggest general functional characterization for 27 more genes (comprising approximately 80% total). Findings from this analysis include the identification of a pyruvate-formate lyase-like operon, likely to be expressed not only in H.influenzae but also in several other bacteria. Further, we also observed three genes that are likely to participate in the transport and/or metabolism of sialic acid, an important component of the H.influenzae lipo-oligosaccharide. Accurate functional annotation of uncharacterized genes calls for an integrative approach, combining expression studies with extensive computational analysis and curation, followed by eventual experimental verification of the computational predictions.

Amino Acid Sequence↗
PubMed2003

Theory of polyelectrolytes in solvents.

Using a continuum description, we account for fluctuations in the ionic solvent surrounding a Gaussian, charged chain and derive an effective short-ranged potential between the charges on the chain. This potential is repulsive at short separations and attractive at longer distances. The chemical potential can be derived from this potential. When the chemical potential is positive, it leads to a meltlike state. For a vanishingly low concentration of segments, this state exhibits scaling behavior for long chains. The Flory exponent characterizing the radius of gyration for long chains is calculated to be approximately 0.63, close to the classical value obtained for second order phase transitions. For short chains, the radius of gyration varies linearly with N, the chain length, and is sensitive to the parameters in the interaction potential. The linear dependence on the chain length N indicates a stiff behavior. The chemical potential associated with this interaction changes sign, when the screening length in the ionic solvent exceeds a critical value. This leads to condensation when the chemical potential is negative. In this state, it is shown using the mean-field approximation that spherical and toroidal condensed shapes can be obtained. The thickness of the toroidal polyelectrolyte is studied as a function of the parameters of the model, such as the ionic screening length. The predictions of this theory should be amenable to experimental verification.

Journal Article↗
PubMed2006

Ganglioside mimicry as a cause of Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS), characterized by acute progressive limb weakness and areflexia, is the prototype of postinfectious autoimmune diseases. Campylobacter jejuni is the most frequently identified agent of infection in GBS patients, often preceding acute motor axonal neuropathy (AMAN), a variant of GBS. Anti-GM1, anti-GM1b, anti-GD1a, and anti-GalNAc-GD1a IgG antibodies are associated with AMAN. Carbohydrate mimicry [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-] was seen between the lipo-oligosaccharide of C. jejuni isolated from an AMAN patient and human GM1 ganglioside. Sensitization with the lipo-oligosaccharide of C. jejuni induces AMAN in rabbits as does sensitization with GM1 ganglioside. Paralyzed rabbits have pathological changes in their peripheral nerves identical to changes seen in human GBS. C. jejuni infection may induce anti-ganglioside antibodies by molecular mimicry, eliciting AMAN. This is the first verification of the causative mechanism of molecular mimicry in an autoimmune disease. To express ganglioside mimics, C. jejuni requires specific gene combinations that function in sialic acid biosynthesis or transfer. The knockout mutants of these landmark genes of GBS show reduced reactivity with GBS patients' sera, and fail to induce an anti-ganglioside antibody response in mice. These genes are crucial for the induction of neuropathogenic cross-reactive antibodies. An approach for evaluating intravenous immune globulin, a treatment for GBS, based on our animal model of AMAN is also discussed in this review, and recent advances made in this field are described.

Animals↗
PubMed2003

[A neurologist's views on cellular and gene therapy in nervous system diseases].

Lesional impairment of the brain and spinal cord is a serious and usually irreversible disorder. There is only a small chance of functionally significant compensation of lesions. Neurons and glia are marked by the minimal regeneration. Though plasticity and adaptability represent important mechanisms, which can contribute to the compensation of structural and functional CNS disorders, irreversible structural and functional deficits may develop. Transplantation of various cells incl. neurons from fetal brain or transplantation of genetically modified cells was therefore proposed. Clear verification of this method by animal tests and by clinically controlled trials is a necessary condition. Its application is expected in atrophic-degenerative and vascular diseases of CNS, tumors, injuries, seizure, inflammatory and demyelinating diseases in which the replacement of neurons, glia and enhancement of their regeneration play the key role. Transplantation of embryonic stem cells, fetal neural cells, and haematopoietic stem cells introduced by stereotaxic, hematogenic or intrathecal procedure gets the attention. Bioethical problems and the possible contribution of this method based on experience with transplantation of fetal cells into basal ganglia of 3 patients with Parkinson's disease and haematopoietic stem cell transplantation in multiple sclerosis (n 1) are discussed.

Animals↗
PubMed1990

Computers in radiotherapy.

Radiotherapy is one of the earliest fields in Medicine in which computers have made an inroad. The main uses of computers, which include treatment planning, dose calculations, localisation of tumours, verification of patient setups and radiation beam data acquisition, are highlighted in this paper. It is believed that a modern Radiotherapy department cannot function optimally without some form of computing facilities. With advances made in dose calculation algorithms, and in high performance computers, it is expected that the target of 3% accuracy dose calculations can be achieved in treatment planning.

Algorithms↗
PubMed1998

Two models of the influenza A M2 channel domain: verification by comparison.

BACKGROUND: The influenza M2 protein is a simple membrane protein, containing a single transmembrane helix. It is representative of a very large family of single-transmembrane helix proteins. The functional protein is a tetramer, with the four transmembrane helices forming a proton-permeable channel across the bilayer. Two independently derived models of the M2 channel domain are compared, in order to assess the success of applying molecular modelling approaches to simple membrane proteins. RESULTS: The Calpha RSMD between the two models is 1.7 A. Both models are composed of a left-handed bundle of helices, with the helices tilted roughly 15 degrees relative to the (presumed) bilayer normal. The two models have similar pore radius profiles, with a pore cavity lined by the Ser31 and Gly34 residues and a pore constriction formed by the ring of His37 residues. CONCLUSIONS: Independent studies of M2 have converged on the same structural model for the channel domain. This model is in agreement with solid state NMR data. In particular, both model and NMR data indicate that the M2 helices are tilted relative to the bilayer normal and form a left-handed bundle. Such convergence suggests that, at least for simple membrane proteins, restraints-directed modelling might yield plausible models worthy of further computational and experimental investigation.

Magnetic Resonance Spectroscopy↗
PubMed2006

Changes in the respiratory pattern during radiotherapy for cancer in the lung.

BACKGROUND AND PURPOSE: To quantify changes in patients' diaphragm motion pattern over the course of radiotherapy and to evaluate the implications of these changes for 4D radiotherapy. PATIENTS AND METHODS: From January 2004 to October 2004, 10 patients with lung malignancies treated at our department underwent weekly respiratory motion verification during the course of external beam radiation. An onboard kilovoltage imaging system was used to acquire fluoroscopy weekly for patients with lung neoplasms. The diaphragm position as a function of time was extracted automatically from the fluoroscopy and used to calculate the daily mean and daily SD of motion. The diaphragm position was related to both a bony reference point and machine isocenter. Changes in the daily mean and daily SD in relation to the reference (first day) daily mean and reference daily SD were measured. RESULTS: The mean change in the daily mean was 0.32 mm+/-6.11 mm in relation to the bony reference point and 0.38 mm+/-6.28 mm in relation to isocenter. The mean change in the daily SD was 0.91 mm+/-1.81 mm. The mean systematic change in the daily mean was 4.97 mm, and the mean random change in the daily mean was 3.61 mm. CONCLUSIONS: Daily verification of 4D radiotherapy techniques to assess the necessity of online set-up correction may be required due to the large change in the mean diaphragm position observed for these patients. However, the variation of the daily SD was small for most patients. Adaptive adjustment of the margin may be necessary for those patients with larger variation of the daily SD.

Adaptation, Physiological↗
PubMed2006

The relationship of inflammatory bowel disease type and activity to psychological functioning and quality of life.

BACKGROUND & AIMS: We aimed to assess the relationship of disease type and disease activity with psychological functioning and quality of life (QOL) in a population-based cohort of patients with recently diagnosed inflammatory bowel disease (IBD). METHODS: A total of 388 individuals diagnosed within 7 years were recruited from a population-based registry of IBD patients for the Manitoba IBD Cohort Study. Participants completed a clinical interview and standardized self-report measures of positive and negative psychological functioning, and QOL. Disease activity was determined by symptom self-report over the prior 6 months; Harvey-Bradshaw or Powell-Tuck disease activity indices also were used. Disease type was determined through chart verification. RESULTS: Seventy-four percent of Crohn's disease and 66% of ulcerative colitis participants had active disease during the previous 6 months. Multivariate regression showed that those with active disease had higher levels of distress, health anxiety, and perceived stress, lower social support, well-being and mastery, and poorer disease-specific QOL, relative to those with inactive disease. Disease type was not contributory to psychological functioning or QOL. Pain anxiety (fear of pain) and pain-specific catastrophizing were not associated with disease activity, after controlling for other psychological variables. Participants with either active or inactive disease had suboptimal general QOL. CONCLUSIONS: Ulcerative colitis and Crohn's disease participants were not differentiated in their psychological profiles. Given the strong association between disease-specific QOL, psychological functioning, and disease activity, it is important to be aware of related difficulties in patients with active IBD. There is a continued impact on QOL by the disease, even when it is inactive.

Adaptation, Psychological↗
PubMed2000

Combined transformation and genetic technique verification of protein-protein interactions in the yeast two-hybrid system.

The yeast two-hybrid system is frequently used to identify protein-protein interactions. The assay is based on the functional reconstitution of a transcriptional activator. Since an indirect phenotype of the positive clones is the basis for selection of positive interacting clones, the two-hybrid screens are vulnerable to false positives. Here we report a screening protocol based on the sequential use of the cotransformation approach followed by the genetic method for verifying true two-hybrid interactions. Using this procedure, we have screened a cDNA library and have been able to isolate true positives from the yeast two-hybrid screen.

Gene Library↗
PubMed1994

Brain potentials reveal deficits of language processing after closed head injury.

OBJECTIVE: To delineate deficits in language processing after closed head injury with use of behavioral measures and event-related brain potentials. DESIGN: Case-control design. All subjects participated in three verbal event-related brain potential experiments, and the resulting measures were compared both within and between groups. PATIENTS/CONTROLS: Eleven patients at least 2 years after severe closed head injury (Glasgow Coma Scale score < 8 at admission and duration of posttraumatic amnesia > 48 hours) were compared with a control group matched for age and educational level. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Reaction times and percentage correct as behavioral measures in the three experiments (sentence verification, semantic and repetition priming with lexical decision task, and continuous word recognition). Event-related brain potentials were quantified by area measures in successive time windows for the different experimental conditions and for different experiments. RESULTS: The reaction times of the patient group were significantly longer than those of the controls (P < .005). Similarly, the patients' accuracy was significantly worse in all experiments (P < .03). The event-related brain potentials of the controls showed a clear and significant reduction of a negative component (N400) to terminal words of true sentences (sentence verification experiment), semantically primed words and repeated words (lexical decision experiment), and recognized words (continuous word recognition). For the patients, a clear N400 effect was seen only in the sentence verification task (delayed by about 100 milliseconds), while only later event-related brain potential modulations were seen in the other tasks. CONCLUSION: Language functions are disturbed after closed head injury. The electrophysiologic data suggest difficulties in the integration of incoming linguistic stimuli with the previous context as a possible underlying cause.

Adolescent↗
PubMed2006

Structural and genetic characterization of Shigella boydii type 17 O antigen and confirmation of two new genes involved in the synthesis of glucolactilic acid.

Shigella strains are human pathogens and normally identified based on their O antigens. The chemical structure and gene cluster of Shigella boydii type 17 O antigen were studied. As judged by sugar and methylation analyses along with NMR spectroscopy data, the O antigen of S. boydii type 17 has a linear trisaccharide O unit, which consists of two residues of N-acetylgalactosamine (GalNAc) and a 4-O-[(R)-1-carboxyethyl]-d-glucose (glucolactilic acid). The O antigen gene cluster of S. boydii type 17 was sequenced and genes encoding UDP-N-acetylglucosamine C4 epimerase for GalNAc synthesis, O unit flippase, O antigen polymerase, and glycosyltransferases were putatively identified based on sequence similarities and the presence of conserved motifs. Two genes, whose functions could not be clearly indicated by homology search, were confirmed to be involved in the synthesis of glucolactilic acid by mutation and structural verification of the O antigens from the mutants. To our knowledge, this is the first time that genes involved in the synthesis of glucolactilic acid have been reported. Two genes specific to S. boydii type 17 were also identified.

Amino Acid Motifs↗
PubMed2004

Social cognitive theory of posttraumatic recovery: the role of perceived self-efficacy.

The present article integrates findings from diverse studies on the generalized role of perceived coping self-efficacy in recovery from different types of traumatic experiences. They include natural disasters, technological catastrophes, terrorist attacks, military combat, and sexual and criminal assaults. The various studies apply multiple controls for diverse sets of potential contributors to posttraumatic recovery. In these different multivariate analyses, perceived coping self-efficacy emerges as a focal mediator of posttraumatic recovery. Verification of its independent contribution to posttraumatic recovery across a wide range of traumas lends support to the centrality of the enabling and protective function of belief in one's capability to exercise some measure of control over traumatic adversity.

Adaptation, Psychological↗