Investigation of the binding preference of reovirus sigma1 for junctional adhesion molecule A by classical and steered molecular dynamics.

Biochemical studies have determined that reoviruses attach to cells by combining attachment protein sigma1 to the binding interface of its receptor protein junctional adhesion molecule A (JAM-A), and the interface normally takes care of the homodimerization of JAM-A. Tighter binding and slower dissociation of for the sigma1-JAM complex than for the JAM-JAM complex have been probed by both biological and atomic force microscopy experiments; however, the mechanism of the binding preference of the attachment protein for JAM-A still remains unclear. With the help of classical and steered molecular dynamics and energy calculations, the unbinding forces and kinetic properties of the complexes are investigated, together with detailed structural information analyses. A multireceptor mechanism is proposed for the binding preference, which can be helpful for future viral infection and vector targeting studies.

Excited-state backbone twisting of polyfluorene as detected from photothermal after-effects.

By means of time-resolved photoluminescence and photothermal techniques, after-effects from excited-state dynamics, energy migration, and conformational rearrangement of poly(9,9-di-n-octyl-2,7-fluorene) (PFO) and its homologues has been examined and interpreted with rotational potential maps from quantum mechanical calculations. Steady-state photoluminescence spectral changes and time-resolved photoluminescence measurements of oligofluorenes and PFO diluted in toluene suggest excited state ring torsion occurring within 30 ps of photoexitation. With all effects from internal conversion/intersystem crossing processes properly accounted for, we show that the conformational changes associated with this twisting motion can be quantitatively probed by means of photothermal methods. Results suggest mean torsion between neighboring fluorene units by ca. 40 degrees upon excitation, in agreement with the shift of rotational potential minimum from +/-40 degrees (and +/-140 degrees) in the ground state to +/-20 degrees (and +/-160 degrees) in the first excited singlet state according to results of quantum mechanical calculations.

Bulbar signs in normal population.

BACKGROUND AND OBJECTIVES: There is lack of published data on bulbar signs among the healthy population. This study aims to determine the range of normality of bulbar signs particularly among the elderly. METHODS: Systemic examination of bulbar signs was carried out according to a predetermined protocol on a cohort of young and elderly healthy subjects. RESULTS: A total of 206 subjects were recruited in the study, 104 young adults with mean age of 20 years, and 102 elderly with mean age of 73 years. Uvula deviation was seen in 28 (26.9%) young subjects and 22 (21.6%) elderly. Irregular tongue border was seen in 17 subjects, unilateral in 4 subjects. Fourteen (6.8%) subjects had deviation on tongue protrusion. Occasional tremor of tongue on protrusion is common in both young and old. Persistent (severe) tongue tremor on protrusion was seen in 18.6% of the elderly, and 4.8% of the young. None of the subjects had tremor of tongue at rest. In gag reflex, absence of gagging response was common in elderly, seen in two thirds of the subjects on stimulation of the posterior pharyngeal wall. However, all the subjects had uvular movement. Habituation or suppression of gagging response was seen in close to 90% of young males. CONCLUSION: There is wide range of normality in bulbar signs in normal population, particularly among the elderly.

Repetitive lumbar punctures as treatment for normal pressure hydrocephalus.

Selected normal pressure hydrocephalus (NPH) patients cannot be treated by shunt operation because of the procedure's high complication rate. We have treated cases in which prolonged clinical improvement of NPH was experienced after one or two lumbar punctures (LPs). We evaluated the predictors of prolonged improvement of NPH symptoms by repeated LP. Thirty-one NPH patients were retrospectively evaluated (age 72.5 +/- 5.8 years). Gait disturbance, urinary incontinence, and cognitive impairment were semiquantified. We divided the patients into three groups (non-responders, temporary responders, and prolonged responders) according to their responses after LP. We analyzed the characteristics of the groups. Gait disturbance (p = 0.046) and urinary incontinence (p = 0.040) scores and total NPH symptom score (p = 0.007) after cerebrospinal fluid drainage were more significantly improved in prolonged responders than in temporary responders. On multiple logistic regression analyses, total NPH score improvement was the only predictor of the prolonged responders (p = 0.03, odds ratio 0.148). Our study showed that some NPH patients could maintain favorable courses for at least 1 year after LP without shunt operation. Repeated LP could be an alternative treatment in selected NPH patients.

Directly monitor protein rearrangement on a nanosecond-to-millisecond time-scale.

In order to directly observe the refolding kinetics from a partially misfolded state to a native state in the bottom of the protein-folding funnel, we used a "caging" strategy to trap the ß-sheet structure of ubiquitin in a misfolded conformation. We used molecular dynamics simulation to generate the cage-induced, misfolded structure and compared the structure of the misfolded ubiquitin with native ubiquitin. Using laser flash irradiation, the cage can be cleaved from the misfolded structure within one nanosecond, and we monitored the refolding kinetics of ubiquitin from this misfolded state to the native state by photoacoustic calorimetry and photothermal beam deflection techniques on nanosecond to millisecond timescales. Our results showed two refolding events in this refolding process. The fast event is shorter than 20 ns and corresponds to the instant collapse of ubiquitin upon cage release initiated by laser irradiation. The slow event is ~60 µs, derived from a structural rearrangement in ß-sheet refolding. The event lasts 10 times longer than the timescale of ß-hairpin formation for short peptides as monitored by temperature jump, suggesting that rearrangement of a ß-sheet structure from a misfolded state to its native state requires more time than ab initio folding of a ß-sheet.

A sensitive malaria immunoperoxidase assay for the detection of Plasmodium falciparum antibody.

A new and rapid malaria immunoperoxidase assay using the enzyme horseradish peroxidase in place of fluorescein isothiocyanate was developed to allow the serological measurement of antimalarial antibody by light microscopy. Acetone-fixed thin blood films prepared from cultured Plasmodium falciparum were used as the source of antigen. This malaria immunoperoxidase assay is as sensitive as, and occasionally more sensitive than, the indirect fluorescent antibody assay. It is easy to perform and the antigen used does not show cross-reactivity with sera from nonmalarial diseases.

Pathologic activity of Plasmodium berghei prevented but not reversed by dexamethasone.

Dexamethasone has recently been shown to block the production of cachectin (implicated in the pathogenesis of cerebral malaria) if administered prior to endotoxin induction of mouse macrophages. Using the hamster cheek pouch-cerebral malaria model, we tested the hypothesis that dexamethasone is effective as a therapeutic agent in severe malaria if given before some yet undefined trigger point in the disease. Infected hamsters were treated with dexamethasone (0.7 mg/kg) daily on days 7-12, 4-12, or 1-12 post-challenge. When treatment was started on day 1, whole body oxygen consumption (used as a measure of erythrocyte transport to sites of diffusion) on day 12 was greater than (P less than 0.05) that of infected control animals, though the degree of anemia was no different in treated and untreated groups. Furthermore, treatment produced a reduction in monocyte accumulation, capillary malfunction, and monocyte/red blood cell aggregate formation observable in the cheek pouch in vivo and a similar reduction in monocyte presence, capillary pathologic change, and multifocal hemorrhage in the brain on postmortem. These data suggest that mediator(s), whose production can be blocked by pretreatment with dexamethasone, are involved in the pathogenesis of disease leading to death of the Plasmodium berghei infected hamster.

Influence of primary tumor site on host anti-tumor immunity.

Stage IV-S neuroblastoma, characterized by a primary tumor plus disseminated tumors in liver, skin and bone marrow, has a favorable clinical prognosis when compared to metastatic Stage IV neuroblastoma. This favorable outcome also characterized mice receiving tumor transplants to these "IV-S" sites. We report the testing of the hypothesis that enhanced anti-tumor immunity in "IV-S" site neuroblastoma recipients explains this improved survival. A million murine C1300 neuroblastoma cells were inoculated into 256 A/J mice to either "IV-S" sites of skin, liver, peritoneal cavity, or to the disseminated stage "IV" sites of subcutaneous tissue, muscle, kidney and lung. After 21 and 28 days of tumor growth, spleen cells from tumor bearing mice were harvested and analyzed by a 51 Cr release lymphocytotoxicity assay. Cytotoxic T cell activity was consistently higher at day 28 than day 21. In the liver and in the peritoneal cavity, cytotoxic T cell activity was higher than in other organs, and at day 28 these values were significantly higher than Stage "IV" sites. On the other hand, skin is not a immunologically privileged site in vivo study.

A computer program in QuickBasic for the selection of tests for the identification of Helicobacter pylori.

Reliable microbiological tests are needed for the identification of bacteria. A program has been written in QuickBasic to identify such tests by using a formula that is based on Gyllenberg's Sum of C(i) and Gyllenberg's Rank R(i). A total of 139 papers on a newly isolated bacterium, Helicobacter pylori, was used as data source for the coding of test results into an input file. The program outputs a list that aids in the determination of suitable tests for the identification of H. pylori. These tests chosen by the formula were found to be correctly identified as supported by later publications on the bacterium.

Detection of surface antigen in Rickettsia tsutsugamushi infected mouse reticuloendothelial cells.

Antibody produced by immunizing CBA/CaJ mice with RE cells from C57B1/6J mice infected 14 days earlier with R. tsutsugamushi Gilliam strain bound readily to Gilliam strain non-cell associated rickettsiae and less readily to the periphery of infected RE cells. Conversely, antibody produced by immunizing with RE cells infected 21 days earlier did not bind to Gilliam rickettsiae but bound to the surface of RE cells from mice infected 21 days earlier. This binding was not related to alloantibodies because these were absorbed prior to testing. The demonstration of rickettsial antibody staining of infected cell associated antigen(s) in this assay system provides a new method for the detection of R. tsutsugamushi infection.

The role of fine needle aspiration for breast cancer in the climacteric--a preliminary report.

Fine needle biopsy has been used with great accuracy in the diagnosis of breast tumours. There have been few reports on its efficacy as an adjunct to other breast screening techniques like x-ray mammography. Abnormal mammograms in the absence of a palpable breast lump are sometimes detected in the climacteric and these pose a problem of tissue sampling for pathologic diagnosis. We have tried to evaluate the technique of radiologically guided fine needle aspiration in such patients. Our report is of an initial series of 13 cases of non-palpable, mammographically abnormal breast lesions. Eight cases were fibroadenoma, 4 cases mammary dysplasia and 1 a benign breast lesion. Representative cytologic samples were obtained in 11 of our 13 cases. Cytologic diagnosis should be correlated with mammographic findings.

Thioflavin T and its photoirradiative derivatives: exploring their spectroscopic properties in the absence and presence of amyloid fibrils.

In this work, we found that, during storage or after UV irradiation, ThT is demethylated or oxidized, forming three derivatives. These three derivatives were purified by high performance liquid chromatography and characterized by mass and nuclear magnetic resonance spectroscopy and the spectroscopic properties of pure ThT and the derivatives carefully compared. Our results show that the emission peak at 450 nm results from oxidized ThT and not from the monomeric form of ThT, as previously proposed. The partial conversion of ThT into oxidized and demethylated derivatives has an effect on amyloid detection using ThT assay. Irradiated ThT has the same lag time as pure ThT in the amyloidogenesis of insulin, but the intensity of the emitted fluorescence is significantly decreased.

Correlates of physical activity level among hemodialysis patients in Selangor, Malaysia.

INTRODUCTION: There is mounting evidence demonstrating the importance of adequate physical activity to promote better well-being among hemodialysis patients. Available data pertaining to the levels of physical activity and its determinants among hemodialysis patients is, however, scarce in Malaysia. The objectives of this study are hence to determine the levels of physical activity and it associated factors among hemodialysis patients. METHODOLOGY: A total of 70 subjects were recruited from three dialysis centres in Selangor. A face-to-face interview was conducted to obtain socio-demographic data and subjects' knowledge on dietary sources. Medical history, biochemical parameters and weight status were obtained from medical records. Physical activity level (PAL) was assessed using the Global Physical Activity Questionnaire (GPAQ). RESULTS: A total of 81.4% and 18.6% of the respondents had low and moderate PALs, respectively. Thus, none of the respondents had high PAL. Serum creatinine, education level, personal income and knowledge score on potassium-related medical complications were factors found to correlate significantly with PAL. Multiple linear regression analysis showed that higher PAL was predicted by a lower knowledge score on dietary sodium source, higher education and higher serum creatinine. CONCLUSION: Despite consistent documentation of the potential positive impact of physical exercise on hemodialysis outcomes, the level of physical activity remains low among these patients. It is hoped that these findings can add to the existing body of knowledge and serve as a supporting document for the formulation of appropriate interventions to improve the status of physical activity among hemodialysis patients in Malaysia.

Mechanistic insights into the physiological functions of cell adhesion proteins using single molecule force spectroscopy.

Intercellular adhesion molecules play an important role in regulating several cellular processes such as a proliferation, migration and differentiation. They also play an important role in regulating solute diffusion across monolayers of cells. The adhesion characteristics of several intercellular adhesion molecules have been studied using various biochemical assays. However, the advent of single molecule force spectroscopy as a powerful tool to analyze the kinetics and strength of protein interactions has provided us with an opportunity to investigate these interactions at the level of a single molecule. The study of interactions involving intercellular adhesion molecules has gained importance because of the fact that qualitative and quantitative changes in these proteins are associated with several disease processes. In this review, we focus on the basic principles, data acquisition and analysis in single molecule force spectroscopy experiments. Furthermore, we discuss the correlation between results obtained using single molecule force experiments and the physiological functions of the proteins in the context of intercellular adhesion molecules. Finally, we summarize some of the diseases associated with changes in intercellular adhesion molecules.

CT planning for breast cancer.

Radiotherapy to the affected breast or chest wall is well established as an integral part of postoperative management of breast cancer. However, it is known to be associated with increased cardiac and pulmonary morbidities and mortalities. Modern technologies, such as CT planning, have shown to improve treatment planning by accurately delivering optimal doses to the target volumes, while minimizing doses to sensitive structures, thus reducing potential treatment-related adverse effects. The purpose of this study is to report on our experiences with CT planning of adjuvant radiotherapy for breast cancer.

Biophysical approaches for studying the integrity and function of tight junctions.

Cell-cell adhesion is an extremely important phenomenon as it influences several biologically important processes such as inflammation, cell migration, proliferation, differentiation and even cancer metastasis. Furthermore, proteins involved in cell-cell adhesion are also important from the perspective of facilitating better drug delivery across epithelia. The adhesion forces imparted by proteins involved in cell-cell adhesion have been the focus of research for sometime. However, with the advent of nanotechnological techniques such as the atomic force microscopy (AFM), we can now quantitatively probe these adhesion forces not only at the cellular but also molecular level. Here, we review the structure and function of tight junction proteins, highlighting some mechanistic studies performed to quantify the adhesion occurring between these proteins and where possible their association with human diseases. In particular, we will highlight two important experimental techniques, namely the micropipette step pressure technique and the AFM that allow us to quantify these adhesion forces at both the cellular and molecular levels, respectively.

Transfer of immunity to cryptococcosis by T-enriched splenic lymphocytes from Cryptococcus neoformans-sensitized mice.

Splenic enriched T-cells and sera were obtained from inbred CBA/J mice injected 7 or 35 days earlier with either 10(3) viable Cryptococcus neoformans or sterile physiological saline. The transfer of enriched T-cells collected 7 days after immunization or of normal enriched T-cells did not transfer immunity to C. neoformans or delayed-type hypersensitivity responsiveness to cryptococcal culture filtrate (CneF) antigen to the recipients. However, enriched T-cells harvested 35 days after immunization, when transferred to recipient mice, were able to confer immunity as indicated by the reduction in numbers of C. neoformans cells in the tissues, and they also transferred delayed-type hypersensitivity responsiveness to CneF antigens. Sera from either sensitized or normal mice were unable to transfer immunity to recipient animals. These results suggested that there was a time requirement for development of the immune response in the donor mice and that T-cells were crucial in the host defense against a cryptococcal infection. Culturing of day-35 C. neoformans-sensitized T-cells in the presence of homologous antigen (CneF) but not in the presence of heterologous antigen (purified protein derivative or 2, 4-dinitro-1-fluorobenzene) induced the production of migration inhibition factor, thus indicating that lymphocytes from C. neoformans-injected mice were specifically sensitized to CneF antigen.

Thymosin fraction 5: effects on T cell functions in mice immunosuppressed by severe dietary protein deficiency.

The present studies were performed to determine the effects of severe protein deficiency and subsequent injection of thymosin fraction 5 (TF5) on T and B cell functions. BALB/c mice, 4 weeks old, were fed a normal protein (21%), a low protein (4%) or a protein free (0%) diet and then injected with TF5 or buffer (PBS). A significant increase was observed in the PHA (phytohemagglutinin) and LPS (lipopolysaccharide) induced mitogenesis with increasing age of the well-nourished, PBS injected animals. The severely protein malnourished mice, PBS injected and the well nourished mice, injected with TF5 had smaller increases in both B and T cell mitogenesis with increasing age. TF5 injection of the malnourished mice increased PHA and LPS mitogenesis nearly to the levels of the well-nourished mice. The protein malnourished mice consistently had higher serum corticosteroid levels than controls. No changes in serum corticosteroids were observed with TF5 injection of controls, but there was a significant decrease in the corticosteroid levels of the severely malnourished with TF5 injection. Cytoxicity assays of T cell function, antibody dependent cellular cytoxicity and cytoxicity to mouse thymona tumor cells, in mice fed moderately protein deficient diets showed suppression compared to controls fed 20% protein. TF5 injection partially and temporarily increased these functions in the malnourished mice.

Suppression of lymphocyte responsiveness during acute Rickettsia tsutsugamushi infection in mice.

Lymphocytes from Inbred Balb/cyJ mice infected subcutaneously with 10(3) MLD50 of Rickettsia tsutsugamushi Karp strain were unresponsive to rickettsial antigens and phytohemagglutinin in the lymphocyte transformation assay between 11 and 28 days post-infection. There was, however, no correlation between this suppressed responsiveness to rickettsial antigens and the immune capacity to resist a normally lethal ip challenge with the Karp strain. Fourteen days after infection, these mice survived a potentially lethal Karp challenge. Naive recipients of day 14 post-infection mouse splenic lymphocytes also were partially protected against ip challenge. Karp strain-specific anti-rickettsial antibody reached a peak geometric mean titer of 260 during the period of apparent cellular unresponsiveness. However, day 14 post infection mice showed a fourfold decrease of antibody response to a T-dependent antigen, which suggests the presence of T-dependent antibody suppression in these mice.