Influenza infections are initiated by the binding of the influenza hemagglutinin (HA) and the cellular receptor sialic acids. The binding is followed by internalization, endocytosis, and uncoating to release the influenza genome to the cytoplasm. It is conceivable that specific inhibitors that antagonize any one of these events could prevent the replication of influenza infections. The authors made HA pseudotyped retroviral vectors that express luciferase reporter activities upon transduction to several recipient cells. The transduction of the HA-pseudotype virus particles (HApp) was mediated through the specific interactions between an avian HA and the terminal disaccharides of sialic acid (SA) and galactose (Gal) in alpha-2,3 linkage. The HApp-mediated transduction method was used to develop a high-throughput screening assay and to screen for hits from a fermentation extract library. Specific hits that inhibited the HA-mediated but were noninhibitory to the vesicular stomatitis virus-mediated pseudoviral transductions were identified. A few of these hits have anti-influenza activities that prevent the replication of both H1N1 (WSN) and H5N1 (RG14) influenza viruses.

OBJECTIVE

The influence of childhood trauma as a specific environmental factor on the development of adult psychopathology is far from being elucidated. As part of a collaborative project between research groups from South Africa (SA) and Sweden focusing on genetic and environmental factors contributing to anxiety disorders, this study specifically investigated rates of childhood trauma in South African and Swedish patients respectively, and whether, in the sample as a whole, different traumatic experiences in childhood are predictive of social anxiety (SAD) or panic disorder (PD) in adulthood.

METHODS

Participants with SAD or PD (85 from SA, 135 from Sweden) completed the Childhood Trauma Questionnaire (CTQ). Logistic regression was performed with data from the two countries separately, and from the sample as a whole, with primary diagnoses as dependent variables, gender, age, and country as covariates, and the CTQ subscale totals as independent variables. The study also investigated the internal consistency (Cronbach alpha) of the CTQ subscales.

RESULTS

SA patients showed higher levels of childhood trauma than Swedish patients. When data from both countries were combined, SAD patients reported higher rates of childhood emotional abuse compared to those with PD. Moreover, emotional abuse in childhood was found to play a predictive role in SAD/PD in adulthood in the Swedish and the combined samples, and the same trend was found in the SA sample. The psychometric qualities of the CTQ subscales were adequate, with the exception of the physical neglect subscale.

CONCLUSIONS

Our findings suggest that anxiety disorder patients may differ across countries in terms of childhood trauma. Certain forms of childhood abuse may contribute specific vulnerability to different types of psychopathology. Longitudinal studies should focus on the potential sequential development of SAD/PD among individuals with childhood emotional abuse.

OBJECTIVE

To test whether there are differences in the levels and ratios of 6 pro- and 3 anti-inflammatory cytokines produced by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) in rheumatoid arthritis (RA) subjects compared to controls.

METHODS

79 participants (42 seropositive RA patients and 37 healthy controls) were enrolled in this study. The production levels in mitogen-stimulated PBMCs of the 6 proinflammatory cytokines (IFN-gamma, TNF-alpha, TNF-beta, IL-8, IL-17, IL-18) and 3 anti-inflammatory cytokines (IL-4, IL-10, IL-13) were assayed by ELISA using kits obtained from Immunotech SA. The ratios of pro- to anti-inflammatory cytokines were calculated for all participants.

RESULTS

There were significantly elevated levels of IL-8 and IL-10, and reduced levels of IFN-gamma, IL-4, and IL-17 in mitogen-stimulated PBMC culture supernatants of RA subjects compared to controls. Of the 18 pro-/anti-inflammatory cytokine ratios, 3 ratios (TNF-alpha/IL13, IL-8/IL-4 and IL-8/IL-13) were significantly higher in RA patients compared to controls; and 6 were higher in controls (IFN-gamma/IL-4; IFN-gamma/IL-10; IFN-gamma/IL-13; TNF-beta/IL10; IL-17/IL-10; IL-18/IL-10).

CONCLUSIONS

Activated PBMCs of RA patients, regardless of disease activity, showed higher-level production of IL-8 and IL-10 compared to controls; lower-level production of IFN-gamma, IL-4, and IL-17; and elevated ratios of TNF-alpha/IL-13, IL-8/IL-4 and IL-8/IL-13.

The ablated particle count and size distribution of four solid matrix materials commonly used for matrix-assisted laser desorption ionization (MALDI) were measured with a scanning mobility particle sizer (SMPS) combined with a light scattering aerodynamic particle sizer (APS). The two particle sizing instruments allowed size measurements in the range from 10 nm to 20 μm. The four solid matrixes investigated were 2,5-dihydroxybenzoic acid (DHB), 4-nitroaniline (NA), α-cyano-4-hydroxycinnamic acid (CHCA), and sinapic acid (SA). A thin film of the matrix was deposited on a stainless steel target using the dried droplet method and was irradiated with a 337 nm nitrogen laser at atmospheric pressure. The target was rotated during the measurement. A large number of nanoparticles were produced, and average particle diameters ranged from 40 to 170 nm depending on the matrix and the laser fluence. These particles are attributed to agglomeration of smaller particles and clusters and/or hydrodynamic sputtering of melted matrix. A coarse particle component of the distribution was observed with diameters between 500 nm and 2 μm. The coarse particles were significantly lower in number but had a total mass that was comparable to that of the nanoparticles. The coarse particles are attributed to matrix melting and spallation. Two of the compounds, CHCA and SA, had a third particle size distribution component in the range of 10 to 30 nm, which is attributed to the direct ejection of clusters.

Mass spectrometry (MS) profiling of the proteome and peptidome for disease-associated patterns is a new concept in clinical diagnostics. The technique, however, is highly sensitive to external sources of variation leading to potentially unacceptable numbers of false positive and false negative results. Before MS profiling can be confidently implemented in a medical setting, standard experimental methods must be developed that minimize technical variance. Past studies of variance have focused largely on pre-analytical variation (i.e., sample collection, handling, etc.). Here, we examined how factors at the analytical stage including the matrix and solid-phase extraction influence MS profiling. Firstly, a standard peptide/protein sample was measured automatically by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS across five consecutive days using two different preparation methods, dried droplet and sample/matrix, of four types of matrix: alpha-cyano-4-hydroxycinnamic acid (HCCA), sinapinic acid (SA), 2,5-dihydroxybenzoic acid (DHB) and 2,5-dihydroxyacetophenone (DHAP). The results indicated that the matrix preparation greatly influenced a number of key parameters of the spectra including repeatability (within-day variability), reproducibility (inter-day variability), resolution, signal strength, background intensity and detectability. Secondly, an investigation into the variance associated with C8 magnetic bead extraction of the standard sample prior to automated MS profiling demonstrated that the process did not adversely affect these same parameters. In fact, the spectra were generally more robust following extraction. Thirdly, the best performing matrix preparations were evaluated using C8 magnetic bead extracted human plasma. We conclude that the DHAP prepared according to the dried-droplet method is the most appropriate matrix to use when performing automated MS profiling.

Based on our initial results on the effects of several ATP-binding cassette (ABC) transporter inhibitors on rhodamine-123 efflux from A549, a human lung carcinoma, and MES-SA, a human uterine sarcoma cell line, the aim of this study was to identify the transporter responsible for this export. Export of two fluorescent dyes, rhodamine-123 and calcein, was investigated in both cell lines by testing five commonly used inhibitors of ABC transporters: verapamil, cyclosporin A, MK571, GF129018 and fumitremorgin C. A very high degree of correlation (R(2)=0.91-0.99) between results obtained in the two cell lines suggested that the same transporter was involved in the export of tested fluorescent substrates in both cell lines. Expression analysis and gene silencing techniques, as well as transport of additional substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDCF) on membrane vesicles revealed that the transporter was multidrug resistance related protein 2 (MRP2, ABCC2). Furthermore, it was found that the tested modulators showed very diverse effects on the export of three fluorescent substrates via MRP2, with some modulators being inhibitory in one, while having no effect or even stimulating the transport in the other fluorescent dye assay. Verapamil inhibited rhodamine-123, but stimulated CDCF transport and did not affect calcein export. GF129018 did not affect calcein and CDCF transport, but it inhibited rhodamine-123 transport. These results demonstrate the importance of studying various combinations of potential substrates and modulators of MRP2 in order to estimate possible drug-drug interactions in living organisms. In addition, A549 and MES-SA cells were shown to be good cell models for studying interactions of compounds with human MRP2.

We report the binding free energy calculation and its decomposition for the complexes of alpha-lytic protease and its protein inhibitors using molecular dynamics simulation. Standard mechanism serine protease inhibitors eglin C and OMTKY3 are known to have strong binding affinity for many serine proteases. Their binding loops have significant similarities, including a common P1 Leu as the main anchor in the binding interface. However, recent experiments demonstrate that the two inhibitors have vastly different affinity towards alpha-lytic protease (ALP), a bacterial serine protease. OMTKY3 inhibits the enzyme much more weakly (by approximately 10(6) times) than eglin C. Moreover, a variant of OMTKY3 with five mutations, OMTKY3M, has been shown to inhibit 10(4) times more strongly than the wild-type inhibitor. The underlying mechanisms for the unusually large difference in binding affinities and the effect of mutation are not well understood. Here we use molecular dynamics simulation with molecular mechanics-Poisson Boltzmann/surface area method (MM-PB/SA) to investigate quantitatively the binding specificity. The calculated absolute binding free energies correctly differentiate the thermodynamic stabilities of these protein complexes, but the magnitudes of the binding affinities are systematically overestimated. Analysis of the binding free energy components provides insights into the molecular mechanism of binding specificity. The large DeltaDeltaG(bind) between eglin C and wild type OMTKY3 towards ALP is mainly attributable to the stronger nonpolar interactions in the ALP-eglin C complex, arising from a higher degree of structural complementarity. Here the electrostatic interaction contributes to a lesser extent. The enhanced inhibition in the penta-mutant OMTKY3M over its wild type is entirely due to an overall improvement in the solvent-mediated electrostatic interactions in the ALP-OMTKY3M complex. The results suggest that for these protein-complexes and similar enzyme-inhibitor systems (1) the binding is driven by nonpolar interactions, opposed by overall electrostatic and solute entropy contributions; (2) binding specificity can be tuned by improving the complementarity in electrostatics between two associating proteins. Binding free energy decomposition into contributions from individual protein residues provides additional detailed information on the structural determinants and subtle conformational changes responsible for the binding specificity.

Spinal anesthesia (SA), accounting for more than 50% of regional anesthesias in the spinal region, is generally perceived as simple and safe. Our purpose is to increase awareness of hemorrhagic complications following SA. A 69-year-old male without either coagulation disorders or anticoagulant/antiplatelet therapy developed acute radiculopathy, and severe mental confusion after SA for prostatectomy. CT showed intracranial subarachnoid and intraventricular acute hemorrhage. Cerebral angiography was negative. MRI showed subarachnoid and subdural hematoma in the dorsolumbar spine. Seven-year follow-up showed permanent cognitive and radicular damage. Multiple attempts for SA most likely caused spinal vessels rupture, either directly or indirectly by inducing differential pressure changes between cerebrospinal fluid and intravascular spaces; however, definite mechanisms have not been completely understood. Patients undergoing spinal puncture must report any neurological abnormality, which may result in irreversible damage. Cases of altered consciousness require an extensive neuroradiological evaluation. Proper competency of physicians responsible for spinal puncture is mandatory.

BACKGROUND

Current interest in the assessment of probing attachment level measurements has been stimulated by recent introduction of novel periodontal probes as well as by the reclassification of periodontal diseases. Clinical attachment level (CAL) is currently the "gold standard" for diagnosing and monitoring periodontal diseases.

OBJECTIVE

To evaluate the performance of the newly introduced cementoenamel junction (CEJ) Probe in detecting CAL using the CEJ as a fixed reference point, and to compare the CEJ Probe with the Florida Disk Probe and a standard Manual Probe (North Carolina Probe).

METHODS

Three examiners probed 12 periodontal patients to determine intra- and inter-consistency of both the probes and the examiners, over a 4-week time interval. Subjects ranged in age from 22 to 74 years. The experimental design was structured to balance the intra- and inter-examiner consistency at the same site during the two visits.

RESULTS

Using the PROC MIXED of SAS, a strong interaction (p<0.001) between the examiner and probes was found. The consistency of probing (repeatability of measurements) depended upon the type of periodontal probe used as well as the skill (experience) of the examiner. Overall, the CEJ Probe displayed a more consistent performance. The CEJ Probe demonstrated greater intra-examiner consistency than the Disk Probe for two examiners (p<0.01). The CEJ Probe also showed increased inter-examiner consistency (p<0.01).

CONCLUSIONS

The CEJ Probe has the potential to offer the dental team an efficient, accurate mechanism to chart and monitor attachment level measurements over time. Additional studies, using large numbers of examiners, are needed to assess more clearly the performance of each individual probe.

Eight species of mushrooms were evaluated for mitogenic activity by the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method using spleen cells of C3H/HeN female mice. The hot water-soluble (HWS) fraction extracted from Sarcodon aspratus showed the highest activity. The mitogen in Sa. aspratus was isolated by Sepharose 6B and DEAE-Sepharose CL-6B column chromatography. Preliminary structural analyses indicated that the mitogen was a fucogalactan. Fucogalactan elicited the release of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in macrophages of mice in vitro. TNF-alpha production induced with 50 microg/ml of fucogalactan was significantly higher than that induced by lentinan (500 microg/ml) by approximately 4.3-fold. Also, fucogalactan showed dose dependence at concentrations from 5 to 500 microg/ml in NO production. Thus, fucogalactan does elicit the release of cytokines such as TNF-alpha and NO.

Site-directed mutagenesis is routinely used in modern biology to elucidate the functional or biophysical roles of protein residues, and plays an important role in the field of rational protein design. Over the past decade, a number of computational tools have been developed that can predict the effect of point mutations on a protein's biophysical characteristics. However, these programs usually provide predictions for only a single characteristic. Furthermore, online versions of these tools are often impractical to use for examination of large and diverse sets of mutants. We have created a new web application, (http://enzyme.ucd.ie/PEAT_SA), that can simultaneously predict the effect of mutations on stability, ligand affinity and pK(a) values. PEAT-SA also provides an expanded feature-set with respect to other online tools which includes the ability to obtain predictions for multiple mutants in one submission. As a result, researchers who use site-directed mutagenesis can access state-of-the-art protein design methods with a fraction of the effort previously required. The results of benchmarking PEAT-SA on standard test-sets demonstrate that its accuracy for all three prediction types compares well to currently available tools. We illustrate PEAT-SA's potential by using it to investigate the influence of mutations on the activity of Subtilisin BPN'. This example demonstrates how the ability to obtain a wide range of information from one source, that can be combined to obtain deeper insight into the influence of mutations, makes PEAT-SA a valuable service to both experimental and computational biologists.

This study of the neural representation of sound location in the bat Pteronotus parnellii describes how the peripheral and central components of its auditory system shape the horizontal and vertical spatial selectivity of single neurons in the inferior colliculus. Pteronotus extracts spatial information from the echoes of an emitted pulse composed of four constant-frequency harmonics (30, 60, 90, and 120 kHz), each terminated by a downward frequency sweep. To quantify the intensity cues available in the echo, cochlear microphonic response thresholds were used to measure the directional selectivity of the ear and the interaural intensity level disparities (IIDs) created between ears at standardized speaker positions in the bat's frontal sound field, at frequencies in the pulse spectrum. Speaker positions where thresholds were lowest were termed the sensitive area (SA) of the ear. Positions where IID values were greater than 10 dB were termed the difference area (DA). Ear directionality exhibited a pronounced frequency dependence, both in terms of the degree of directional selectivity and the position of the SA. At the 30-kHz harmonic of the pulse, the ear was broadly directional; the SA covered most of the lower half of the ipsilateral field. The ear was highly directional at the 60- and 90-kHz harmonics. Also, the vertical position of the SA changed dramatically between 60 and 90 kHz, from the horizontal midline at 60 kHz to 40 degrees below the midline at 90 kHz. The positions of the DAs also showed a pronounced frequency dependence. The 30-kHz DA was restricted to the extreme lateral part of the frontal sound field. The 60- and 90-kHz DAs were located in the same positions as the equivalent SAs and exhibited the same difference in vertical position. The DAs of the pulse harmonics differ in both their horizontal and vertical positions; the ears thus generate pronounced binaural spectral cues, which provide two-dimensional spatial information. In the inferior colliculus, a combined paradigm of closed-field dichotic stimulation, followed by free-field stimulation, was used to document the frequency tuning and binaural response properties of single neurons and to correlate these properties with the neuron's horizontal and vertical spatial selectivity in the frontal sound field. Where a neuron responded to free-field stimulation at the lowest intensity is termed its SA. A neuron's frequency tuning primarily influenced its degree of spatial selectivity and its sensitivity in the vertical plane, reflecting the directional properties of the external ears at the neuron's best frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

A new chlorosulfonylated tetradentate beta-diketone, 4,4'-bis(1",1",1",2",2",3",3"-heptafluoro-4",6"-hexanedion-6 "-yl) chlorosulfo-o-terphenyl (BHHCT), was synthesized as a chelating label for Eu3+. BHHCT can be covalently bound to proteins under mild conditions and forms a strongly fluorescent chelate with Eu3+. Bovine serum albumin (BSA) and streptavidin (SA) were labeled with BHHCH-Eu3+, and the latter was used for time-resolved fluoroimmunoassay of alpha-fetoprotein (AFP) in human sera. A remarkably high sensitivity was obtained, with a detection limit of 4.1 x 10(-3) pg/mL, which corresponds to an improvement of about 4-5 orders of magnitude, compared to those of all conventional immunoassays including fluoroimmunoassay, enzyme immunoassay, and radioimmunoassay. The high sensitivity has been attained both by strong fluorescence of the present label and by the extremely suppressed background level brought about by the direct labeling of proteins with the beta-diketone-Eu3+ complex. A general consideration and ideas are given for designing ideal label ligands for strongly fluorescent Eu3+ complexes.

Greater acculturation has been linked to increased risk of cardiovascular disease in Hispanics. C-reactive protein (CRP), a marker of inflammation, is known to be associated with an increased risk of cardiovascular disease morbidity and mortality. Whether acculturation is associated with CRP levels in Hispanics has not been established. We examined the association between acculturation and CRP in 11,858 Hispanic-American adults participating in the National Health and Nutrition Examination Survey (NHANES) from 1999 to 2008. Acculturation was measured by the Short Acculturation Scale (SAS), a validated language-based scale. We used multivariate linear regression to examine the independent association between acculturation and CRP after adjusting for clinical and demographic covariates and appropriate sampling weights. We back-transformed the beta coefficients into relative differences (RDs). Higher acculturation was independently associated with higher CRP levels in Hispanics. Compared to those less acculturated, the RD in CRP levels was 52% higher (p = 0.003) for more acculturated Hispanics. Other significant predictors of CRP in Hispanics included a higher body mass index (RD 139% higher per 5 kg/m(2)), female gender (RD 36% higher), education level (RD 19% higher levels for at least a high school education, p <0.001), being insured (RD 27% higher CRP level, p = 0.006), having hypertension (RD 40% higher CRP levels, p <0.001), and statin use (RD 22% lower CRP levels, p = 0.002). In conclusion, higher acculturation was associated with increased CRP levels in Hispanics in a nationally representative population survey. Inflammation may play an important role in explaining the association between acculturation and increased cardiovascular risk.

BACKGROUND

Hemagglutination (HA) and hemagglutination inhibition (HI) assays are conventionally used for detection and identification of influenza viruses. HI assay is also used for detection of antibodies against influenza viruses. Primarily turkey or chicken erythrocytes [red blood cells (RBCs)] are used in these assays, as they are large, nucleated, and sediment fast, which makes it easy to determine the titer. Human influenza viruses agglutinate RBCs from chicken, human, and guinea pig, but not from horse. Human influenza viruses bind preferentially to sialic acid (SA) linked to galactose (Gal) by α 2, 6 linkage (SA α 2, 6-Gal), whereas avian influenza (AI) viruses bind preferentially to SA α 2, 3-Gal linkages. With this background, the present study was undertaken to study erythrocyte binding preferences and receptor specificities of AI viruses isolated from India.

METHODS

A total of nine AI virus isolates (four subtypes) from India and three reference AI strains (three subtypes) were tested in HA and HI assays against mammalian and avian erythrocytes. The erythrocytes from turkey, chicken, goose, guinea pig and horse were used in the study. The receptor specificity determination assays were performed using goose and turkey RBCs. The amino acids present at 190 helix, 130 and 220 loops of the receptor-binding domain of the hemagglutinin protein were analyzed to correlate amino acid changes with the receptor specificity.

RESULTS

All tested highly pathogenic avian influenza (HPAI) H5N1 viruses reacted with all five types of RBCs in the HA assay; AI H9N2 and H5N2 viruses did not react with horse RBCs. For H5N1 viruses guinea pig and goose RBCs were best for both HA and HI assays. For H9N2 viruses, guinea pig, fowl and turkey RBCs were suitable. For other tested AI subtypes, avian and guinea pig RBCs were better. Eight isolates of H5N1, one H4N6 and one H7N1 virus showed preference to avian sialic acid receptors. Importantly, two isolates of HPAI H5N1, H9N2 and H11N1 viruses showed receptor specificity preference to both avian and mammalian sialic acid (α-2, 3 and α-2, 6) receptors.

CONCLUSIONS

Use of different types of RBCs resulted in titer variations in HA and HI assays. This showed that RBCs giving optimum HA and HI titers would increase sensitivity of detection and would be more appropriate for identification and antigenic analysis of AI viruses. Analysis of 16 amino acids in the receptor-binding domain of the hemagglutinin of HPAI H5N1 viruses revealed that the only variation observed was in S221P amino acid position. Two H5N1 viruses showed S221P amino acid change, out of which only one H5N1 virus showed preference to α 2, 6 sialic acid receptor. One H5N1 virus isolate with amino acid S at 221 position, showed preference to α 2,3 as well as α 2,6 sialic acid receptors. This indicated that factor(s) other than S221P mutation in the hemagglutinin are probably involved in determining receptor specificity of H5N1 viruses. This is the first report of receptor specificity and erythrocyte binding preferences of AI viruses from India.

The aim of the present study was to investigate the association between the serum lipid profile and components of the metabolic syndrome, such as central obesity (anthropometric, computed tomography and fat cell data), insulin, sex-hormone-binding-globulin (SHBG) and different hormones influencing this important syndrome, e.g. sex steroids, leptin and tumor necrosis factor-alpha (TNF-alpha). The sample consisted of 85 obese patients (30 men and 55 women) who had undergone abdominal surgery. Fasting serum lipids were analysed, as well as anthropometric and computed tomography data, perivisceral and subcutaneous fat cell size and serum glucose and hormones. Abdominal fat revealed itself as an important correlator of the adverse changes in plasma lipoprotein levels, the waist-to-hip-ratio and waist-to-thigh-ratio being the best morphological correlators in men and women, respectively. Intra-abdominal fat (VA) correlated significantly and positively to perivisceral fat cell size in women, while no correlation was found between subcutaneous fat accumulation (SA) and adipocyte size in both genders. Perivisceral fat cell size showed the greatest number of correlations with the adverse plasma lipid profile compared to that in the subcutaneous depot. SHBG and sex steroids showed a negative correlation with serum lipids considered a cardiovascular risk. In contrast, TNF-alpha and C-peptide were inversely correlated with potential protector lipids. In conclusion, abdominal obesity, adipocyte hypertrophy from visceral fat, serum TNF-alpha and C-peptide seem to be the best correlators of the lipoprotein disturbance characteristic of the metabolic syndrome, whereas SHBG and sex steroids could play a protective role regarding the lipid profile associated to this syndrome.

Large-diameter elastic arteries can produce strong contractions indefinitely at a high-energy economy by the formation of latch bridges. Whether downstream blood vessels also use latch bridges remains unknown. The zero-pressure medial thickness and lumen diameter of rabbit saphenous artery (SA), a muscular branch of the elastic femoral artery (FA), were, respectively, approximately twofold and half-fold that of the FA. In isolated FA and SA rings, KCl rapidly (< 16 s) caused strong increases in isometric stress (1.2 x 10(5) N/m2) and intracellular Caation ([Caaapproximately 175 nM in both tissues, but stress was sustained in FA (1.3 x 10(5) N/m2) and reduced by 40% in SA (0.8 x 10(5) N/m2). Reduced tonic stress correlated with reduced myosin light chain (MLC) phosphorylation in SA (28 vs. 42% in FA), and simulations with the use of the four-state kinetic latch-bridge model supported the hypothesis that latch-bridge formation in FA, but not SA, permitted maintenance of high stress values at steady state. SA expressed more MLC phosphatase than FA, and permeabilized SA relaxed more rapidly than FA, suggesting that MLC phosphatase activity was greater in SA than in FA. The ratio of fast-to-slow myosin isoforms was greater for SA than FA, and on quick release, SA redeveloped isometric force faster than FA. These data support the hypothesis that maintained isometric force was 40% less in SA than in FA because expressed motor proteins in SA do not support latch-bridge formation.

The porcine serum inhibitor alpha 2-macroglobulin prevents influenza virus from entering host cells by competing for the SA alpha 2, 6Gal-binding site of the hemagglutinin (HA). We studied a series of inhibitor-sensitive and inhibitor-resistant human and porcine influenza virus isolates of the H3N2 subtype, all of which contained HAs, which initially bound only to SA alpha 2, 6Gal oligosaccharides. When their neuraminidase was inhibited, the naturally resistant viruses, as a result of no longer being able to elute from the inhibitor, became sensitive. Evidently it is the neuraminidase which enabled these viruses to grow in hosts which possess the inhibitor. Escape-mutants selected under laboratory conditions in the presence of porcine serum became inhibitor-resistant by two alternative mechanisms: they changed either their HA-specificity or their neuraminidase-specificity. The study thus disclosed two evolutionary strategies for acquiring resistance to a host neuraminidase-sensitive inhibitor: (i) acquisition of an HA able to bind to oligosaccharides not present on the inhibitor; or (ii) acquisition of a neuraminidase able to cleave the oligosaccharide bound by the HA.

Aberrant glycosylation of serum IgA1 was considered as an initial event and involvement in the pathogenesis of IgAN. We previously demonstrated that aberrant glycosylation of serum IgA1 was associated with pathologic phenotype of IgAN. The present study is to investigate if abnormal sialylation of IgA1 affects renal survival of IgAN. 127 patients with biopsy-proven IgAN were enrolled and followed up to 8 years. Seventy-nine healthy and 75 patients with non-IgAN renal diseases were selected as controls. Alpha 2, 6 sialic acid (SA) of serum IgA1 was measured by sandwich-ELISA. Renal survival rate was estimated by Kaplan-Meier method. Alpha 2, 6 SA level in patients with IgAN was lower than that in healthy controls (0.92+/-0.14 vs. 0.98+/-0.12, P=0.001) and non-IgAN glomerulonephritis (0.92+/-0.14 vs. 1.00+/-0.18, n=53, P=0.001). Patients with IgAN in Low SA Group were no significant differences compared with patients in Normal SA Group in age, gender, hypertension, serum creatinine, and excretion of proteinuria. Renal cumulative survival rate was 53.3% in patients in Low SA Group and 83.5% in Normal SA Group (P=0.0008). The lower the alpha 2, 6 SA level of serum IgA1 in patients with IgAN was, the worse their renal survival rate was. Although patients in Low SA Group had worse renal function evaluated by eGFR, there was no significant difference in various CKD stages in non-IgAN renal function controls (n=42, P=0.352). Alpha 2, 6 SA level of serum IgA1 was associated with the prognosis of patients with IgAN and could serve as a predictor of poor prognosis in IgAN.

The aim of this study was to investigate symptoms of social anxiety and the psychometric properties of the Social Anxiety Scale for Adolescents (SAS-A) among Finnish adolescents, 13-16 years of age. Study 1 (n = 867) examined the distribution of SAS-A scores according to gender and age, and the internal consistency and factor structure of the SAS-A. In a subsample (n = 563; Study 2) concurrent and discriminant validity of the SAS-A were examined relative to the Social Phobia Inventory and the Beck Depression Inventory. Test-retest stability was examined over a 30-month period by repeated measures every 6 months in another subsample (n = 377; Study 3). Results mostly revealed no gender differences in social anxiety, except that boys reported more general social avoidance and distress than girls. Older adolescents (14-16-year-olds) reported higher social anxiety than younger adolescents (12-13-year-olds). Internal consistency for the SAS-A was acceptable for both genders and for all three SAS-A subscales. Confirmatory factor analysis replicated the original 18-item three-factor structure of the SAS-A, accounting for 61% of the variance between items. Evidence for concurrent and discriminant validity was found. Test-retest stability over 6 months was satisfactory. Results support the reliability and validity of the Finnish adaptation of the SAS-A, and further indicate that gender differences in adolescents' social anxiety may vary across Western countries.

The expression of PR-1a gene in tobacco is accompanied by changes in the chromatin architecture over its promoter region. The transcription initiates when the gene is induced in defense response, a condition that can be simulated experimentally by external application of salicylic acid. Mutagenesis of the core promoter sequence established that the TATA-box was critical to the expression of PR-1a gene. In order to study functional specificity between the core promoter and upstream activator region, the native core promoter was exchanged with that of a heterologous salicylic acid inducible promoter, Pcec. The core promoter and the activator region of PR-1a together determine its tightly regulated expression, slow kinetics of induction by SA and several fold induction of expression. In uninduced state, a single nucleosome was present over the core promoter of PR-1a. It masked both the TATA-box and the transcription initiation region. The transcriptional activation of the promoter by SA was accompanied by shift in the position of this nucleosome. The chimeric promoters failed to show inducibility or gave very low level of induction. They showed failure in shifting the nucleosome from the core promoter region. The promoter Pcec expressed constitutively at a high uninduced level in spite of a nucleosome over the TATA-box region. However, in this case, the nucleosome did not mask the transcript initiation region. The TATA-box nucleosome was shifted as the expression increased further, following induction by SA. A fully induced Pcec had the TATA-box fully exposed, though a weak nucleosome appeared on the +1 region. The results support a close relationship among promoter sequence architecture, nucleosome positioning and PR-1a expression.

The present study was undertaken to evaluate the antioxidant efficacy of alpha-eleostearic acid and punicic acid, two isomers of conjugated linolenic acid, in terms of normalization of altered biochemical parameters of oxidative stress following sodium arsenite treatment in rats. Animals were divided into four groups. The first group used as control. While, group 2, 3 and 4 were orally treated with alpha-eleostearic acid (0.5% of total lipid given) plus sodium arsenite (Sa; 10mg/kgBW), punicic acid (0.5% of total lipid given) plus sodium arsenite (Sa; 10mg/kg BW) and sodium arsenite (Sa; 10mg/kg BW), respectively. Results showed that activities of antioxidant enzymes decreased significantly due to oxidative stress generated by sodium arsenite. Lipid peroxidation also increased due to sodium arsenite administration. alpha-Eleostearic acid and punicic acid acted as antioxidant and caused mostly all the altered parameters restored to normal level. Results also showed that antioxidant activity of alpha-eleostearic acid was more predominant than that of punicic acid.

BACKGROUND

Combination therapy in managing psychiatric disorders is not uncommon. While combination therapy has been documented for depression and schizophrenia, little is known about combination therapy practices in managing attention-deficit/hyperactivity disorder (ADHD). This study seeks to quantify the combination use of ADHD medications and to understand predictors of combination therapy.

METHODS

Prescription dispensing events were drawn from a U.S. national claims database including over 80 managed-care plans. Patients studied were age 18 or over with at least 1 medical claim with a diagnosis of ADHD (International Classification of Diseases, Ninth Revision, Clinical Modification [ICD-9-CM] code 314.0), a pharmacy claim for ADHD medication during the study period July 2003 to June 2004, and continuous enrollment 6 months prior to and throughout the study period. Dispensing events were grouped into 6 categories: atomoxetine (ATX), long-acting stimulants (LAS), intermediate-acting stimulants (IAS), short-acting stimulants (SAS), bupropion (BUP), and Alpha-2 Adrenergic Agonists (A2A). Events were assigned to calendar months, and months with combined use from multiple categories within patient were identified. Predictors of combination therapy for LAS and for ATX were modeled for patients covered by commercial plans using logistic regression in a generalized estimating equations framework to adjust for within-patient correlation between months of observation. Factors included age, gender, presence of the hyperactive component of ADHD, prior diagnoses for psychiatric disorders, claims history of recent psychiatric visit, insurance plan type, and geographic region.

RESULTS

There were 18,609 patients identified representing a total of 11,886 months of therapy with ATX; 40,949 months with LAS; 13,622 months with IAS; 38,141 months with SAS; 22,087 months with BUP; and 1,916 months with A2A. Combination therapy was present in 19.7% of continuing months (months after the first month of therapy) for ATX, 21.0% for LAS, 27.4% for IAS, 23.1% for SAS, 36.9% for BUP, and 53.0% for A2A.For patients receiving LAS, being age 25-44 or age 45 and older versus being 18-24 years old, seeing a psychiatrist, having comorbid depression, or having point-of-service coverage versus a Health Maintenance Organization (HMO) resulted in odds ratios significantly greater than 1, representing increased likelihood for combination therapy in managing adult ADHD.For patients receiving ATX, being age 25-44 or age 45 and older versus being 18-24 years old, seeing a psychiatrist, having a hyperactive component to ADHD, or having comorbid depression resulted in odds ratios significantly greater than 1, representing increased likelihood for combination therapy in managing adult ADHD.

CONCLUSIONS

ATX and LAS are the most likely drugs to be used as monotherapy. Factors predicting combination use were similar for months in which ATX was used and for months in which LAS was used except that a hyperactive component to ADHD predicted increased combination use for ATX but not for LAS.

The thermal stability of several commonly used crystalline matrix-assisted ultraviolet laser desorption/ionization mass spectrometry (UV-MALDI-MS) matrices, 2,5-dihydroxybenzoic acid (gentisic acid; GA), 2,4,6-trihydroxyacetophenone (THA), alpha-cyano-4-hydroxycinnamic acid (CHC), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid; SA), 9H-pirido[3,4-b]indole (nor-harmane; nor-Ho), 1-methyl-9H-pirido[3,4-b]indole (harmane; Ho), perchlorate of nor-harmanonium ([nor-Ho+H]+) and perchlorate of harmanonium ([Ho+H]+) was studied by heating them at their melting point and characterizing the remaining material by using different MS techniques [electron ionization mass spectrometry (EI-MS), ultraviolet laserdesorption/ionization-time-of-flight-mass spectrometry (UV-LDI-TOF-MS) and electrospray ionization-time-of-flight-mass spectrometry (ESI-TOF-MS)] as well as by thin layer chromatography analysis (TLC), electronic spectroscopy (UV-absorption, fluorescence emission and excitation spectroscopy) and 1H nuclear magnetic resonance spectroscopy (1H-NMR). In general, all compounds, except for CHC and SA, remained unchanged after fusion. CHC showed loss of CO2, yielding the trans-/cis-4-hydroxyphenylacrilonitrile mixture. This mixture was unambiguously characterized by MS and 1H-NMR spectroscopy, and its sublimation capability was demonstrated. These results explain the well-known cluster formation, fading (vanishing) and further recovering of CHC when used as a matrix in UV-MALDI-MS. Commercial SA (SA 98%; trans-SA/cis-SA 5:1) showed mainly cis- to-trans thermal isomerization and, with very poor yield, loss of CO2, yielding (3',5'-dimethoxy-4'-hydroxyphenyl)-1-ethene as the decarboxilated product. These thermal conversions would not drastically affect its behavior as a UV-MALDI matrix as happens in the case of CHC. Complementary studies of the photochemical stability of these matrices in solid state were also conducted.