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X-linked Myopathy with Excessive Autophagy (XMEA) is a childhood onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p, VMA21 is an essential assembly chaperone of the vacuolar ATPase (V-ATPase), the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids which leads to downregulation of the mTORC1 pathway, and consequent increased macroautophagy resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge, and vacuolate the cell. Our results uncover a novel mechanism of disease, namely macroautophagic overcompensation leading to cell vacuolation and tissue atrophy.

Aim: To determine if visual acuity is tested reliably in primary health care in New Zealand., Methods: Fifteen to 26 'eyes' from seven participants were tested in the Eye Department of Dunedin Hospital under standardised conditions; and across 17 centres in nine general practices and the Emergency Department of Dunedin Hospital for comparison. Variables including lighting and distance were measured; chart type and centre conditions were recorded., Results: Eleven centres (65%) produced visual acuity scores that were inconsistent with the Eye Department, where 10 (59%) of them produced worse visual acuity scores and one centre (6%) produced better visual acuity score. Ten centres (59%) did not meet New Zealand Transport Agency standards of adequate illumination of greater than 500 lux. Ten centres (59%) failed to have their charts at the specified distance., Conclusion: There were inconsistencies in visual acuity testing in primary health care in Dunedin, New Zealand which may be related to the overall poor compliance with lighting and distance standards. These factors are potentially easily modifiable and their change should lead to improvements in visual acuity measurements.

The progressive myoclonus epilepsies (PMEs) consist of a group of diseases with myoclonic seizures and progressive neurodegeneration, with onset in childhood and/or adolescence. Lafora disease is a neuronal glycogenosis in which normal glycogen is transformed into starch-like polyglucosans that accumulate in the neuronal somatodendritic compartment. It is caused by defects of two genes of yet unknown function, one encoding a glycogen phosphatase (laforin) and the other an ubiquitin E3 ligase (malin). Early cognitive deterioration, visual seizures affecting over half, and slowing down of EEG basic activity are three major diagnostic clues. Unverricht-Lundborg disease is presently thought to be due to damage to neurons by lysosomal cathepsins and reactive oxygen species due to absence of cystatin B, a small protein that inactivates cathepsins and, by ways yet unknown, quenches damaging redox compounds. Preserved cognition and background EEG activity, action myoclonus early morning and vertex spikes in REM sleep are the diagnostic clues. Sialidosis, with cherry-red spot, neuronopathic Gaucher disease, with paralysis of verticality, and ataxia-PME, with ataxia at onset in the middle of the first decade, are also lysosomal diseases. How the lysosomal defect culminates in myoclonus and epilepsy in these conditions remains unknown., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Hypothesis: The optimal timing for surgical repair of the supraspinatus (SSP) tendon after full-substance tear has not been established. The objectives of this prospective investigation of SSP tendon repair delayed by 1, 2, or 3 months followed by a 3-month postoperative course were to (1) determine the site of failure, (2) measure the tensile strength and stiffness, and (3) assess the ability of computed tomography to predict mechanical strength., Materials and Methods: We transected 1 SSP tendon in 36 rabbits and then repaired it with transosseous sutures after a delay of 1, 2, or 3 months. We compared the results with 36 intact shoulders from 18 age-matched control rabbits., Results: Experimental specimens failed at the tendon (n = 26) more often than at the enthesis (n = 10) (P < .05). The mean peak loads to failure 3 months after repair delayed by 1 month and delayed by 2 months were significantly greater than their respective control values (P < .05 for both); there was no difference after a delay of 3 months. There was no association between the presence of hypoattenuation on computed tomography and repair strength (P > .05)., Conclusions: Our findings indicate better mechanical results with earlier repair (1 or 2 months) after SSP tendon than after a delay of 3 months. Early surgical repair may lower the risk of tendon retear., (Copyright © 2012 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Mosby, Inc. All rights reserved.)

Objective: The purpose of this study was to establish the contemporaneous frequency of metastases within the kidney as opposed to primary renal tumors in patients with an active primary nonrenal malignancy and to identify the differentiating features., Materials and Methods: We retrospectively identified all patients with an active primary nonrenal malignancy (group 1) who had also undergone at least 2 contrast-enhanced abdominal CT examinations spaced 1 year apart. The radiologic and pathologic data of these cases were reviewed and the incidence of metastasis to the kidney versus primary renal tumors established. These data were compared with a separate group who presented with primary renal malignancy from the outset (group 2)., Results: In the study were 2340 patients with primary nonrenal malignancy (group 1) and 231 patients with a primary renal malignancy (group 2). For group 1, the mean age was 63 years and 51% were men; for group 2, the mean age was 59 years, and 58% were men. The differences were not statistically significant. Thirty-six patients in group 1 had a malignant renal mass; 21 were a result of kidney metastasis and the remaining 15 were a synchronous primary renal tumor (0.9% vs 0.6%). The kidney was the eighth most common site of metastatic spread. Metastases to the kidney were statistically more likely with higher tumor stage of the primary nonrenal malignancy (68% vs 46%, p = 0.0006) and in those with other sites of metastasis (p = 0.012, positive likelihood ratio [LR+] = 6.75). Compared with primary renal tumors, metastases to the kidney were more often solid (86% vs 53%, p = 0.019, LR+ = 3.7) and endophytic (76% vs 33%, p = 0.017, LR+ = 2.29). There were too few cases with calcification and bilateral tumors to reach a statistically significant conclusion. Tumor size, polar predominance, and enhancement pattern were similar in the two groups. The primary renal tumors seen in group 1 versus group 2 were similar regarding age and sex distribution, cell type, median size, and tumor stage., Conclusion: Metastases to the kidney are uncommon in modern radiologic practice (0.9%, 21/2340 in this study), and a renal mass seen in a patient with nonrenal malignancy is nearly as likely to be an incidental primary renal tumor. Metastasis is more likely in those with higher tumor stage or if other viscera are also affected and is usually an asymptomatic, small, endophytic, and solid mass. If a renal mass seen in a patient with primary nonrenal malignancy proves to be a synchronous primary renal tumor, its cell type and stage will be similar to sporadic primary renal tumors.

Cancer patients spontaneously generate autoantibodies (AAb) to tumor-derived proteins. To detect AAb, we have probed novel high-density custom protein microarrays (NAPPA) expressing 4988 candidate tumor antigens with sera from patients with early stage breast cancer (IBC), and bound IgG was measured. We used a three-phase serial screening approach. First, a prescreen was performed to eliminate uninformative antigens. Sera from stage I-III IBC (n = 53) and healthy women (n = 53) were screened for AAb to all 4988 protein antigens. Antigens were selected if the 95th percentile of signal of cases and controls were significantly different (p < 0.05) and if the number of cases with signals above the 95th percentile of controls was significant (p < 0.05). These 761 antigens were screened using an independent set of IBC sera (n = 51) and sera from women with benign breast disease (BBD) (n = 39). From these, 119 antigens had a partial area under the ROC curve (p < 0.05), with sensitivities ranging from 9-40% at >91% specificity. Twenty-eight of these antigens were confirmed using an independent serum cohort (n = 51 cases/38 controls, p < 0.05). Using all 28 AAb, a classifier was identified with a sensitivity of 80.8% and a specificity of 61.6% (AUC = 0.756). These are potential biomarkers for the early detection of breast cancer.

Recurrence of tears is a common complication after rotator cuff surgery. Retearing seems to occur early after surgery and may be attributed to too early or too vigorous exercises. We found no experimental data correlating the strength of the rotator cuff early after surgery and imaging. Our objectives were to measure the peak load to failure of rabbit supraspinatus tendon-bone constructs at early times postoperatively, to determine their mode of failure, and to determine whether computed tomography (CT) can predict their strength. We divided one supraspinatus tendon of 40 adult female white New Zealand rabbits and, after resection of the enthesis, sutured the tendon into a bony trough. Ten rabbits were killed immediately and 10 each at 1, 2, and 6 weeks postoperatively. The explanted tendons of both shoulders were imaged on CT and tested to failure. Compared to normal tendons (mean 210 +/- 42 N), the mean strength was very low at 0 weeks (57 +/- 21 N) and 1 week (86 +/- 33 N) (both p < 0.05); it had recovered by 6 weeks (324 +/- 66 N). Early on, suture pullout was the most common mode of failure, whereas at 6 weeks, mid-substance tears predominated (p < 0.05). Hypoattenuation on CT was associated with increased strength of the tendon-bone construct (p < 0.05). The strength of the surgical construct is very low in the early postoperative period. Therefore, the shoulder should be submitted only to loads not interfering with healing., ((c) 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

No vaccine exists for the prevention of infection with the ubiquitous gastric pathogen Helicobacter pylori, and drug therapy for the infection is complicated by poor patient compliance, the high cost of treatment, and ineffectiveness against drug-resistant strains. A new medical advancement is required to reduce the incidence of peptic ulcer disease and stomach cancer, two conditions caused by infection with H. pylori. Clinical trials have been performed with a formalin-inactivated H. pylori whole cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of Escherichia coli heat-labile toxin. Following the initial dose of this vaccine, some subjects experienced gastrointestinal side effects. To reduce side effects and potentially further increase the amount of adjuvant that can safely be administered with the HWC vaccine, experiments were performed with a form of LT that carried two mutations in the A subunit, a substitution of G for R at position 192, and A for L at position 211. The double mutant LT (dmLT) adjuvant stimulated immune responses as effectively as the single mutant LT in mice. Additionally, following a challenge infection, the dmLT-adjuvanted vaccine was as effective as single mutant LT in reducing gastric urease levels (diagnostic for H. pylori infection), and H. pylori colonization in the stomach as assessed by quantitative analysis of stomach homogenates. A lyophilized formulation of HWC was developed to improve stability and to potentially reduce reliance on cold chain maintenance. It was observed that a dmLT-adjuvanted lyophilized vaccine was equally as protective in the mouse model as the liquid formulation as assessed by gastric urease analysis and analysis of stomach homogenates for viable H. pylori. No readily detectable effect of tonicity or moisture content was observed for the lyophilized vaccine within the formulation limits evaluated. In an accelerated stability study performed at 37 degrees C the lyophilized vaccine remained equally as protective as vaccine stored at 2-8 degrees C. The formulation selected for clinical development consisted of 2.5 x 10(10) formalin-inactivated cells per ml in 6.5% trehalose, 0.5% mannitol, and 10mM citrate buffer at pH 6.8., ((c) 2009 Elsevier Ltd. All rights reserved.)

Purpose: To determine the usefulness of clinical imaging in predicting the mechanical properties of rabbit Achilles tendons after acute injury., Methods: We created a 2 x 7-mm full-thickness central tendon defect in one Achilles tendon of healthy rabbits. Rabbits in groups of 10 were killed immediately and 4 and 8 wk after surgery (n = 30). We then performed magnetic resonance (MR) imaging, ultrasonography (US), bone mineral densitometry (BMD), and mechanical testing to failure using a dual-cryofixation assembly on experimental and contralateral tendons. The main outcome measures included tendon dimensions, optical density (OD) of T1-weighted, proton density (PD), and T2-weighted MR sequences, US focal abnormalities, BMD of the calcaneus, and stress and peak load to failure., Results: On MR imaging and US, all dimensions of the injured tendons after 2 wk and more were greater than those of the contralateral tendons (P < 0.05). The mean T1-weighted OD was greater at 4 wk (256 +/- 53) and 8 wk (184 +/- 24) than immediately after surgery (149 +/- 15). Mechanical stress was markedly lower in the experimental than in the contralateral tendons at both 4 wk (39 +/- 9 vs 77 +/- 16 N x mm(-2)) and 8 wk (58 +/- 6 vs 94 +/- 26 N x mm(-2); P < 0.05). Mean peak load to failure was significantly lower immediately after surgery (332 +/- 128 N) than at 4 and 8 wk (712 +/- 106 and 836 +/- 90 N, respectively). Both high T1-weighted OD (r = -0.73) and PD OD (r = -0.69) correlated with lower mechanical stress (P < 0.05). In the experimental tendons, higher T1-weighted OD correlated with lower peak load (r = -0.46; P < 0.05)., Conclusions: Normal peak loads 4 wk after injury were withstood by an enlarged tendon of lower stress. These findings support progressive physical loading 4 wk after an Achilles tendon injury. T1-weighted OD constituted a marker of tendon mechanical recovery.

Immobility in bed and decreased mobility cause adaptations to most human body systems. The effect of immobility on fat accumulation in hemopoietic bone marrow has never been measured prospectively. The reversibility of marrow fat accumulation and the effects on hemopoiesis are not known. In the present study, 24 healthy women (age: 25-40 yr) underwent -6 degrees head-down bed rest for 60 days. We used MRI to noninvasively measure the lumbar vertebral fat fraction at various time points. We also measured hemoglobin, erythropoietin, reticulocytes, leukocytes, platelet count, peripheral fat mass, leptin, cortisol, and C-reactive protein during bed rest and for 1 yr after bed rest ended. Compared with baseline, the mean (+/-SE) fat fraction was increased after 60 days of bed rest (+2.5+/-1.1%, P<0.05); the increase persisted 1 yr after the resumption of regular activities (+2.3+/-0.8%, P<0.05). Mean hemoglobin levels were significantly decreased 6 days after bed rest ended (-1.36+/-0.20 g/dl, P<0.05) but had recovered at 1 yr, with significantly lower mean circulating erythropoietin levels (-3.8+/-1.2 mU/ml, P<0.05). Mean numbers of neutrophils and lymphocytes remained significantly elevated at 1 yr (+617+/-218 neutrophils/microl and +498+/-112 lymphocytes/microl, both P<0.05). These results constitute direct evidence that bed rest irreversibly accelerated fat accumulation in hemopoietic bone marrow. The 2.5% increase in fat fraction after 60 days of bed rest was 25-fold larger than expected from historical ambulatory controls. Sixty days of bed rest accelerated by 4 yr the normal bone marrow involution. Bed rest and marrow adiposity were associated with hemopoietic stimulation. One year after subjects returned to normal activities, hemoglobin levels were maintained, with 43% lower circulating erythropoietin levels, and leukocytes remained significantly elevated across lineages. Lack of mobility alters hemopoiesis, possibly through marrow fat accumulation, with potentially wide-ranging clinical consequences.

Objective: To assess the value of ultrasonography (US), magnetic resonance imaging (MRI), and bone mineral densitometry (BMD) in evaluating human Achilles' tendon strength., Design: Cross-sectional observational study., Setting: Tertiary care hospital., Participants: Ninety-eight Achilles' tendons from 49 consecutive cadavers (26 men and 23 women with a mean age of 66.6 years) undergoing hospital autopsy were assessed., Interventions: Not applicable., Main Outcome Measures: Tendon dimensions on US and MRI, and T1-weighted optical density were measured. Areas of hypodensity, hyperdensity, calcification, and heterogeneity were identified on US. The BMD of each calcaneus was recorded. The tendons were mechanically tested to determine peak load at failure., Results: Sixteen patients (32.7%, 27 tendons) had abnormalities in 1 or both tendons on US and/or MRI (17 on US, 17 on MRI). Fifty-seven tendons (58%) ruptured in their midsubstance, at an average peak load of 4722+/-990N. Tendons with and without abnormalities on imaging had similar strengths (P>.05). Calcaneal BMD correlated weakly with peak load at failure (r=.21, P<.05)., Conclusions: The prevalence of Achilles' tendons abnormalities on US or MRI was 32.7% in our study group. Abnormalities on clinical imaging (US or MRI) were not predictive of the load at failure. Therefore, tendons with imaging abnormalities are not necessarily weaker, and one cannot predict the likelihood of rupture based on imaging results. Further, higher-powered studies could explore the ability of BMD to detect minimal clinically important differences and to predict Achilles' tendon weakness.

Autosomal recessively inherited progressive myoclonus epilepsies (PMEs) include Lafora disease, Unverricht-Lundborg disease, the neuronal ceroid lipofuscinoses, type I sialidosis (cherry-red spot myoclonus), action myoclonus-renal failure syndrome, and type III Gaucher disease. Almost all the autosomal recessively inherited PMEs are lysosomal diseases, with the exception of Lafora disease in which neither the accumulating material nor the gene products are in lysosomes. Progress in identifying the causative defects of PME is near-complete. Much work lies ahead to resolve the pathobiology and neurophysiology of this group of devastating disorders.

X-linked myopathy with excessive autophagy (XMEA) is a childhood-onset disease characterized by progressive vacuolation and atrophy of skeletal muscle. We show that XMEA is caused by hypomorphic alleles of the VMA21 gene, that VMA21 is the diverged human ortholog of the yeast Vma21p protein, and that like Vma21p it is an essential assembly chaperone of the V-ATPase, the principal mammalian proton pump complex. Decreased VMA21 raises lysosomal pH, which reduces lysosomal degradative ability and blocks autophagy. This reduces cellular free amino acids, which upregulates the mTOR pathway and mTOR-dependent macroautophagy, resulting in proliferation of large and ineffective autolysosomes that engulf sections of cytoplasm, merge together, and vacuolate the cell. Our results uncover macroautophagic overcompensation leading to cell vacuolation and tissue atrophy as a mechanism of disease.

The search for new biomarkers for diagnosis, prognosis, and therapeutic monitoring of diseases continues in earnest despite dwindling success at finding novel reliable markers. Some of the current markers in clinical use do not provide optimal sensitivity and specificity, with the prostate cancer antigen (PSA) being one of many such examples. The emergence of proteomic techniques and systems approaches to study disease pathophysiology has rekindled the quest for new biomarkers. In particular the use of protein microarrays has surged as a powerful tool for large-scale testing of biological samples. Approximately half the reports on protein microarrays have been published in the last two years especially in the area of biomarker discovery. In this review, we will discuss the application of protein microarray technologies that offer unique opportunities to find novel biomarkers., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The humoral immune response is a highly specific and adaptive sensor for changes in the body's protein milieu, which responds to novel structures of both foreign and self antigens. Although Igs represent a major component of human serum and are vital to survival, little is known about the response specificity and determinants that govern the human immunome. Historically, antigen-specific humoral immunity has been investigated using individually produced and purified target proteins, a labor-intensive process that has limited the number of antigens that have been studied. Here, we present the development of methods for applying self-assembling protein microarrays and a related method for producing 96-well formatted macroarrays for monitoring the humoral response at the proteome scale. Using plasmids encoding full-length cDNAs for over 850 human proteins and 1700 pathogen proteins, we demonstrate that these microarrays are highly sensitive, specific, reproducible, and can simultaneously measure immunity to thousands of proteins without a priori protein purification. Using this approach, we demonstrate the detection of humoral immunity to known and novel self-antigens, cancer antigens, autoimmune antigens, as well as pathogen-derived antigens. This represents a powerful and versatile tool for monitoring the immunome in health and disease., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel method of inducing the delivery of nonpermeant molecules to the cytosol of cells is presented in this paper. Corona discharge in air was utilized to produce ions that in turn were deposited onto the liquid surface of media containing cultured cells. Murine B16 melanoma cells were used to demonstrate the molecular delivery of fluorescent dye calcein, the drug bleomycin, and a nucleic acid stain SYTOX-green. None of these molecules penetrate cells with intact membranes. Following the corona treatment, cells were observed to admit significant quantities of these molecules from the culture media, relative to control samples. Further, greater than 95% viability of treated cells was observed by Trypan Blue assay. This method may provide an attractive alternative to electroporation where a physical contact between electrodes and cells is needed to deliver molecules to the cytosol.

We developed a high-density self-assembling protein microarray, based on the nucleic acid programmable protein array (NAPPA) concept, to display thousands of proteins that are produced and captured in situ from immobilized cDNA templates. We arrayed up to 1,000 unique human cDNAs and obtained high yields of protein expression and capture with minimal variation and good reproducibility. This method will enable various experimental approaches to study protein function in high throughput.

The wide availability of multidetector row computed tomography (MDCT) continues to change the way we manage patients. Once the domain of the specialist paediatric centre, imaging of the paediatric thorax now routinely occurs in many general hospitals. The paediatric respiratory and radiology teams should therefore be familiar with this technique. This review examines the indications for MDCT, the different types of scan, the use of low-dose protocols for paediatric imaging and appropriate reconstruction techniques. Patient preparation, with particular respect to sedation and optimal contrast bolus delivery, is also addressed.

There is strong preclinical evidence that cancer, including breast cancer, undergoes immune surveillance. This continual monitoring, by both the innate and the adaptive immune systems, recognizes changes in protein expression, mutation, folding, glycosylation, and degradation. Local immune responses to tumor antigens are amplified in draining lymph nodes, and then enter the systemic circulation. The antibody response to tumor antigens, such as p53 protein, are robust, stable, and easily detected in serum; may exist in greater concentrations than their cognate antigens; and are potential highly specific biomarkers for cancer. However, antibodies have limited sensitivities as single analytes, and differences in protein purification and assay characteristics have limited their clinical application. For example, p53 autoantibodies in the sera are highly specific for cancer patients, but are only detected in the sera of 10-20% of patients with breast cancer. Detection of p53 autoantibodies is dependent on tumor burden, p53 mutation, rapidly decreases with effective therapy, but is relatively independent of breast cancer subtype. Although antibodies to hundreds of other tumor antigens have been identified in the sera of breast cancer patients, very little is known about the specificity and clinical impact of the antibody immune repertoire to breast cancer. Recent advances in proteomic technologies have the potential for rapid identification of immune response signatures for breast cancer diagnosis and monitoring. We have adapted programmable protein microarrays for the specific detection of autoantibodies in breast cancer. Here, we present the first demonstration of the application of programmable protein microarray ELISAs for the rapid identification of breast cancer autoantibodies.

Malaria caused by protozoan parasites belonging to the genus Plasmodium is a dreaded disease, second only to tuberculosis. The emergence of parasites resistant to commonly used drugs and the lack of availability of vaccines aggravates the problem. One of the preventive approaches targets adhesion of parasites to host cells and tissues. Adhesion of parasites is mediated by proteins called adhesins. Abrogation of adhesion by either immunizing the host with adhesins or inhibiting the interaction using structural analogs of host cell receptors holds the potential to develop novel preventive strategies. The availability of complete genome sequence offers new opportunities for identifying adhesin and adhesin-like proteins. Development of computational algorithms can simplify this task and accelerate experimental characterization of the predicted adhesins from complete genomes. A curated positive dataset of experimentally known adhesins from Plasmodium species was prepared by careful examination of literature reports. "Controversial" or "hypothetical" adhesins were excluded. The negative dataset consisted of proteins representing various intracellular functions including information processing, metabolism, and interface (transporters). We did not include proteins likely to be on the surface with unknown adhesin properties or which are linked even indirectly to the adhesion process in either of the training sets. A nonhomology-based approach using 420 compositional properties of amino acid dipeptide and multiplet frequencies was used to develop MAAP Web server with Support Vector Machine (SVM) model classifier as its engine for the prediction of malarial adhesins and adhesin-like proteins. The MAAP engine has six SVM classifier models identified through an exhaustive search from 728 kernel parameters set. These models displayed an efficiency (Mathews correlation coefficient) of 0.860-0.967. The final prediction P(maap) score is the maximum score attained by a given sequence in any of the six models. The results of MAAP runs on complete proteomes of Plasmodium species revealed that in Plasmodium falciparum at P(maap) scores above 0.0, we observed a sensitivity of 100% with two false positives. In P. vivax and P. yoelii an optimal threshold P(maap) score of 0.7 was optimal with very few false positives (upto 5). Several new predictions were obtained. This list includes hypothetical protein PF14&#95;0040, interspersed repeat antigen, STEVOR, liver stage antigen, SURFIN, RIFIN, stevor (3D7-stevorT3-2), mature parasite-infected erythrocyte surface antigen or P. falciparum erythrocyte membrane protein 2, merozoite surface protein 6 in P. falciparum, circumsporozoite proteins, microneme protein-1, Vir18, Vir12-like, Vir12, Vir18-like, Vir18-related and Vir4 in P. vivax, circumsporozoite protein/thrombospondin related anonymous proteins, 28 kDa ookinete surface protein, yir1, and yir4 of P. yoelii. Among these, a few proteins identified by MAAP were matched with those identified by other groups using different experimental and theoretical strategies. Most other interspersed repeat proteins in Plasmodium species had lower P(maap) scores. These new predictions could serve as new leads for further experimental characterization (MAAP webserver: http://maap.igib.res.in)., ((c) 2007 Wiley-Liss, Inc.)

We report the production and availability of over 7000 fully sequence verified plasmid ORF clones representing over 3400 unique human genes. These ORF clones were derived using the human MGC collection as template and were produced in two formats: with and without stop codons. Thus, this collection supports the production of either native protein or proteins with fusion tags added to either or both ends. The template clones used to generate this collection were enriched in three ways. First, gene redundancy was removed. Second, clones were selected to represent the best available GenBank reference sequence. Finally, a literature-based software tool was used to evaluate the list of target genes to ensure that it broadly reflected biomedical research interests. The target gene list was compared with 4000 human diseases and over 8500 biological and chemical MeSH classes in approximately 15 Million publications recorded in PubMed at the time of analysis. The outcome of this analysis revealed that relative to the genome and the MGC collection, this collection is enriched for the presence of genes with published associations with a wide range of diseases and biomedical terms without displaying a particular bias towards any single disease or concept. Thus, this collection is likely to be a powerful resource for researchers who wish to study protein function in a set of genes with documented biomedical significance.

Emerging evidence indicates the importance of G-quadruplex motifs as drug targets. [Stuart A. Borman, Ascent of quadruplexes-nucleic acid structures become promising drug targets. Chem. Eng. News, 2007;85, 12-17], which stems from the fact that these motifs are present in a surprising number of promoters wherein their role in controlling gene expression has been demonstrated for a few. We present a compendium of quadruplex motifs, with particular focus on their occurrence and conservation in promoters-QuadBase. It is composed of two parts (EuQuad and ProQuad). EuQuad gives information on quadruplex motifs present within 10 kb of transcription starts sites in 99 980 human, chimpanzee, rat and mouse genes. ProQuad contains quadruplex information of 146 prokaryotes. Apart from gene-specific searches for quadruplex motifs, QuadBase has a number of other modules. 'Orthologs Analysis' queries for conserved motifs across species based on a selected reference organism; 'Pattern Search' can be used to fetch specific motifs of interest from a selected organism using user-defined criteria for quadruplex motifs, i.e. stem, loop size, etc. 'Pattern Finder' tool can search for motifs in any given sequence. QuadBase is freely available to users from non-profit organization at http://quadbase.igib.res.in/.

Objective: To assess the usefulness of magnetic resonance imaging (MRI), ultrasound (US) imaging, or bone mineral density (BMD) in predicting the mechanical properties of immobilized rabbit Achilles' tendons., Design: Experimental study., Setting: Basic university laboratory., Animals: Twenty-eight rabbits., Interventions: Twelve rabbits had 1 hindlimb casted for 4 weeks and 10 rabbits were casted for 8 weeks. Contralateral legs and 12 normal hindlimbs served as controls., Main Outcome Measures: Achilles' tendon dimensions on MRI and US, T1- and T2-signal intensities on MRI, classification of abnormalities on MRI and US; BMD of the calcaneus with dual-energy x-ray absorptiometry. Biomechanic measures consisted of peak load, stiffness, and stress. Imaging variables were correlated with biomechanic alterations., Results: Immobilized Achilles' tendons were weaker and showed decreased mechanical stress compared with their contralateral legs and controls (all P<.05). MRI and US revealed larger Achilles' tendons after immobilization. However, neither increased MRI nor US signal abnormality was found. BMD was lower in immobilized calcanei and larger in contralateral legs than controls. Only BMD correlated with both the decreased peak load (R2=.42, P<.05) and stress (R2=.54, P<.05) of immobilized Achilles' tendon., Conclusions: This study established weakened mechanical properties of immobilized Achilles' tendons. BMD of the calcaneus, but not MRI and US, was predictive of the mechanical alterations in immobilized Achilles' tendons. BMD may be a useful biomarker to monitor disease and recovery in Achilles' tendons.

Motifs that are evolutionarily conserved in proteins are crucial to their structure and function. In one of our earlier studies, we demonstrated that the conserved motifs occurring invariantly across several organisms could act as structural determinants of the proteins. We observed the abundance of glycyl residues in these invariantly conserved motifs. The role of glycyl residues in highly conserved motifs has not been studied extensively. Thus, it would be interesting to examine the structural perturbations induced by mutation in these conserved glycyl sites. In this work, we selected a representative set of invariant signature (IS) peptides for which both the PDB structure and mutation information was available. We thoroughly analyzed the conformational features of the glycyl sites and their local interactions with the surrounding residues. Using Ramachandran angles, we showed that the glycyl residues occurring in these IS peptides, which have undergone mutation, occurred more often in the L-disallowed as compared with the L-allowed region of the Ramachandran plot. Short range contacts around the mutation site were analyzed to study the steric effects. With the results obtained from our analysis, we hypothesize that any change of activity arising because of such mutations must be attributed to the long-range interaction(s) of the new residue if the glycyl residue in the IS peptide occurred in the L-allowed region of the Ramachandran plot. However, the mutation of those conserved glycyl residues that occurred in the L-disallowed region of the Ramachandran plot might lead to an altered activity of the protein as a result of an altered conformation of the backbone in the immediate vicinity of the glycyl residue, in addition to long range effects arising from the long side chains of the new residue. Thus, the loss of activity because of mutation in the conserved glycyl site might either relate to long range interactions or to local perturbations around the site depending upon the conformational preference of the glycyl residue., ((c) 2007 Wiley-Liss, Inc.)

The purpose of this study was to assess the effect of elective bilateral femoral arterial punctures for uterine artery embolization (UAE) of symptomatic fibroids on fluoroscopy and procedural time, patient dose, and ease of procedure. We conducted a prospective study of UAE with either the intention to catheterize both uterine arteries using a single femoral puncture (n = 12) or elective bilateral arterial punctures from the outset (n = 12). The same two operators undertook each case. Main outcome measures were total procedure time, fluoroscopy time, dose-area product (DAP), and total skin dose. A simulation was then performed on an anthropomorphic phantom using the mean in vivo fluoroscopy parameters to estimate the ovarian dose. Bilateral UAE was achieved in all patients. None of the patients with initial unilateral arterial puncture required further contralateral arterial puncture. The mean fluoroscopy time in the group with elective bilateral punctures was 12.8 min, compared with a mean of 16.6 min in patients with unilateral puncture (p = 0.046). There was no significant difference in overall procedure time (p = 0.68). No puncture-site complications were found. Additional catheters were required only following unilateral puncture. The simulated dose was 25% higher with unilateral puncture. Although there was no significant difference in measured in vivo patient dose between the two groups (DAP, p = 0.32), this is likely to reflect the wide variation in other patient characteristics. Allowing for the small study size, our results show that the use of elective bilateral arterial punctures reduces fluoroscopy time, requires less catheter manipulation, and, according to the simulation model, has the potential to reduce patient dose. The overall procedure time, however, is not significantly reduced.

High and broad transcription of eukaryotic genes is facilitated by cost minimization, clustered localization in the genome, elevated G+C content, and low nucleosome formation potential. In this scenario, illumination of correlation between abundance of (TG/CA)(n>or=12) repeats, which are negative cis modulators of transcription, and transcriptional levels and other commonly occurring dinucleotide repeats, is required. Three independent microarray datasets were used to examine the correlation of (TG/CA)(n>or=12) and other dinucleotide repeats with gene expression. Compared with the expected equi-distribution pattern under neutral model, highly transcribed genes were poor in repeats, and conversely, weakly transcribed genes were rich in repeats. Furthermore, the inverse correlation between repeat abundance and transcriptional levels appears to be a global phenomenon encompassing all genes regardless of their breadth of transcription. This selective pattern of exclusion of (TG/CA)(n>or=12) and (AT)(n>or=12) repeats in highly transcribed genes is an additional factor along with cost minimization and elevated GC, and therefore, multiple factors govern high transcription of genes. We observed that even after controlling for the effects of GC and average intron lengths, the effect of repeats albeit somewhat weaker was persistent and definite. In the ribosomal protein coding genes, sequence analysis of orthologs suggests that negative selection for repeats perhaps occurred early in evolution. These observations suggest that negative selection of (TG/CA)(n>or=12) microsatellites in the evolution of the highly expressed genes was also controlled by gene function in addition to intron length.

Functional classification of proteins is central to comparative genomics. The need for algorithms tuned to enable integrative interpretation of analytical data is felt globally. The availability of a general,automated software with built-in flexibility will significantly aid this activity. We have prepared ARC (Automated Resource Classifier), which is an open source software meeting the user requirements of flexibility. The default classification scheme based on keyword match is agglomerative and directs entries into any of the 7 basic non-overlapping functional classes: Cell wall, Cell membrane and Transporters (C), Cell division (D), Information (I), Translocation (L), Metabolism (M), Stress(R), Signal and communication (S) and 2 ancillary classes: Others (O) and Hypothetical (H). The keyword library of ARC was built serially by first drawing keywords from Bacillus subtilis and Escherichia coli K12. In subsequent steps,this library was further enriched by collecting terms from archaeal representative Archaeoglobus fulgidus, Gene Ontology, and Gene Symbols. ARC is 94.04% successful on 6,75,663 annotated proteins from 348 prokaryotes. Three examples are provided to illuminate the current perspectives on mycobacterial physiology and costs of proteins in 333 prokaryotes. ARC is available at http://arc.igib.res.in.

Osteopoikilosis is a rare, inherited bone disorder, which is usually found incidentally on x ray. It may be mistaken for other, more serious disorders such as bony metastases, causing undue distress to the doctor and patient.

PEST sequences are one of the major motifs that serve as signal for the protein degradation and are also involved in various cellular processes such as phosphorylation and protein-protein interaction. In our earlier study, we found that these motifs contribute largely to eukaryotic protein disorder. This observation led us to evaluate their conformational variability in the nonredundant Protein Data Bank (PDB) structures. For this purpose, crystallographic temperature factors, structural alignment of multiple NMR models, and dihedral angle order parameters have been used in this study. The study has revealed the hypermobility of PEST motifs as compared to other regions of the protein. Conformational flexibility may allow them to participate in number of molecular interactions under different conditions. This analysis may explain the role of protein backbone flexibility in bringing about multiple cellular roles of PEST motifs., (2006 Wiley-Liss, Inc.)

This investigation studies porcine tissue response in tooth extraction sockets treated with root replicas made out of Beta-tricalcium phosphate (Beta-TCP; Beta-Ca(3)(PO(4))(2)) granules, molded and held together by thermal fusion of a thin film of polyglycolic-polylactic acid copolymer. Six left mandibular third incisors (n (1)/4 6) of experimental pigs are treated with the root replicas and four contralateral incisors are used as nontreated controls (n (1)/4 4). Two animals each were killed at 20, 40, and 60 weeks of observation periods. The mandibular jaw segments were prepared in toto for light microscopy by resin embedding and serial ground sectioning. Additionally, one Beta-TCP-treated socket at 60 weeks was thoroughly investigated by correlative light, electron microscopic and electron probe X-ray microanalysis to assess the bio-absorbability and host removal of the replica material from the implant site. The extraction wounds of the animals healed satisfactorily with very little histologically observable differences in the healing pattern of the test and control sites. The Beta-TCP was completely removed from extracellular sites, but at 60 weeks, remnants of it were found in the cytoplasm of multinucleated giant cells. The root replicas made out of Beta-TCP were biocompatible and bioabsorbable. Osseous healing occurred both in the test and control sockets, but the healing process was delayed due to the presence of Beta-TCP particles.

In contemporary practice, most prostate cancers are either invisible on ultrasound or indistinguishable from concurrent benign prostatic hyperplasia. Diagnosis therefore rests on prostate biopsy. Biopsies are not simply directed at ultrasonically visible lesions, as these would miss many cancers; rather the whole gland is sampled. The sampling itself is systematic, using patterns based on prostate zonal anatomy and the geographical distribution and frequency of cancer. This review explains the evolution of the prostate biopsy technique, from the classical sextant biopsy method to the more recent extended biopsy protocols (8, 10, 12, >12 and saturation biopsy protocols). Extended protocols are increasingly being used to improve diagnostic accuracy, especially in those patients who require repeat biopsy. This trend has been facilitated by the ongoing improvement in safety and acceptability of the procedure, particularly with the use of antibiotic prophylaxis and local anaesthesia. The technical details of these extended protocols are discussed, as are the current data regarding procedure-related morbidity and how this may be minimized.

Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throughput production and printing of proteins, a laborious method that raises concerns about the stability of the proteins and the shelf life of the arrays. A novel method of producing protein microarrays, called nucleic acid programmable protein array (NAPPA), overcomes these limitations by synthesizing proteins in situ. NAPPA entails spotting plasmid DNA encoding the relevant proteins, which are then simultaneously transcribed and translated by a cell-free system. The expressed proteins are captured and oriented at the site of expression by a capture reagent that targets a fusion protein on either the N- or C-terminus of the protein. Using a mammalian extract, NAPPA expresses and captures 1000-fold more protein per feature than conventional protein-printing arrays. Moreover, this approach minimizes concerns about protein stability and integrity, because proteins are produced just in time for assaying. NAPPA has already proven to be a robust tool for protein functional assays.

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.

Mechanical testing of soft musculoskeletal structures like tendons and ligaments are essential to medical advances. A long-standing limitation for testing these structures in isolation has been the ability to solidly fix both ends of the tendon. Cryogenic fixation technology was leveraged into the development of a dual cryogenic fixation (DCF) device. Results of the study show that the DCF allows tendons to be tested in isolation, at physiologic temperatures, with excellent reproducibility.

Tooth eruption across the mucosa in humans has been studied rarely, although there are disturbances of eruption that are attributed specifically to failure of the supraosseous eruptive migration. The aim of this study was to analyze the soft tissues covering normally erupting teeth so as to get an insight into the supraosseous phase of tooth eruption and to provide the basis for comparison with cases of eruption disturbances. Six opercula covering normally erupting permanent molars (primary opercula) and six of succedaneous teeth (secondary opercula) were surgically removed from 10 patients aged 7.5-17.5 years. Specimens were examined light and electron microscopically and analyzed morphometrically. All opercula contained strands and islands of odontogenic epithelium, prominent numbers of high endothelial venules, nerves, and mast cells. Nerves comprised normally structured, 1.5-3.5 microm thick myelinated (Adelta) and thinner unmyelinated (C) fibers. In primary opercula, the proportions of blood vessels and nerves were three- and sevenfold higher than the respective values for the secondary opercula. Furthermore, primary opercula contained multinucleated, fibroblast-like giant cells that were not observed in secondary opercula. As all teeth under investigation were erupting normally, neither the presence of the giant cells nor the atypical proportions of blood vessels and nerves appeared to be decisive in the eruption process. These conspicuous tissue components of opercula seem merely to accompany the eruptive tooth movement., (Copyright 2005 Wiley-Liss, Inc.)

Quantitative variation in gene expression in humans is the outcome of various factors, including differences in genetic background, gender, age, and environment. However, the extent of the influence of these factors on gene expression is not clear. We attempted to address this issue by carrying out gene expression profiling in blood leukocytes with 13 individuals (including 5 pairs of monozygotic twins) on 10,000 genes using HG-U95Av2 oligonucleotide microarrays. The proportion of differentially expressed genes between monozygotic twins was low (up to 1.76%). Most of the variations belonged to the least variable category. These genes, exhibiting "random variations," did not show clear preference to any functional class, although "signaling and communication" and "immune and related functions" generally topped the list. The extent of variation in gene expression increased in comparisons between unrelated individuals (up to 14.13%). Most of the genes (89%) exhibiting random variations in twins also varied in expression in unrelated individuals. As with twins, signaling and communication topped the list, and substantial variations were observed in all three categories: least variable, moderately variable, and most variable. An important outcome of this study was that the housekeeping genes were nearly insensitive to random variations but appeared to be more susceptible to genetic differences. However, the highly expressed housekeeping genes exhibited low variation and appeared to be insensitive to all known factors. Gene expression profiling in monozygotic twins can provide useful data for the assessment of natural variation in gene expression in humans.

Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays.

The use of magnetic fluids and magnetic particles in combinatorial hyperthermia therapy for cancer treatment is reviewed. The investigation approach adopted for producing thermoregulating particles and tissue model studies for studying particle retention and heating characteristics is discussed.

Mortality patterns of two Zebu cattle breeds, Sahiwal and Tharparkar, and two crossbred strains, Karan Swiss and Karan Fries, maintained at the National Dairy Research Institute, Karnal were studied. Nine-year (1989--90 to 1997--98) data on mortality were analysed for year, season, age and cause effects on mortality rate. The overall mortality was 14.17%. The mortality in Sahiwal, Tharparkar, Karan Swiss and Karan Fries averaged 14.35%, 7.21%, 17.12% and 13.46%, respectively. The breed mortality rate did not vary significantly between years, seasons, age categories and causes of disease. However, the trends indicated appreciable difference in mortality rates. The mortality was highest in the year 1994--95 (19.53%) and lowest in 1991--92 (8.56%). There was very little variation in seasonal mortality rate and mortality rate averaged 4.53%, 4.81% and 4.84% in hot-dry (March-June), hot-humid (July-October) and cold (November-February) seasons, respectively. The mortality up to 2 months of age accounted for a major share (50-60% or higher) in different breed groups. Digestive problems followed by respiratory disorders together accounted for 70-80% of total deaths.

Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

Objective: The objective was to conduct a clinical, radiographic, and histologic follow-up of alveolar socket healing in 8 human cases in which the extraction sockets of the involved teeth were treated with biodegradable root replicas before metallic implants were placed., Study Design: Chair side prepared solid and porous forms of root replicas made out of polylactic-polyglycolic acids (PLGA) copolymer were utilized. Five patients were treated with the solid form and 3 with the porous form of the replicas. The cases were followed up at regular intervals postoperatively, and standardized photographs and radiographs were taken. The cylindrical core of biopsies that were removed with trephine for placement of titanium implants were processed and examined by light and transmission-electron microscopy., Results: Both forms of the root replicas were well tolerated and biodegraded by the body. There were no histologically observable pathological tissue reactions at the time of implant application. However, the solid form seemed to cause an initial decalcification of the bone surrounding the extraction sockets that was subsequently repaired along with the bone healing of the extraction sockets. Such initial decalcification of the alveolar process was not observed in the cases that were treated with the porous form of root replicas. There was wide variation in the osseous component of the trephine-harvested biopsies in both treatment groups that suggests inconsistency in bone healing of the alveolar sockets., Conclusion: The 2 forms of root replicas under investigation were found to be biocompatible and biodegradable. But the compact solid form may cause an initial temporary lactic acid induced decalcification of the alveolar process, which makes it unsuitable for regular clinical application as compared to the granular porous form. The observed inconsistent and unpredictable bone regeneration calls for further research to develop more optimal replica materials.

The unravelling of human genome sequence gives a new opportunity to investigate the role of repetitive sequences in gene regulation. Among the various types of repetitive sequences, the dinucleotide (TG:CA)(n) repeats are one of the most abundant in human genome and exhibit polymorphism. Early on, it was observed that the (TG:CA)(n) repeats could modulate gene expression and has the propensity to undergo conformational transitions in in vivo conditions. Recent reports describe the role of polymorphic (TG:CA)(n) repeats in gene regulation in several genes. In this work, we have analysed the distribution of (TG:CA)(n) (n >or= 6) repeats in human 'housekeeping genes' on which recently released Gene Chip data is available. Our results indicate that (i). The number of short intragenic (TG:CA)(n) repeats is significantly higher than the number of long repeats (ii). the proportion of genes with (TG:CA)(n) repeats (n >or= 12 units) had lower mean expression levels compared to those without these repeats, (iii). the genes belonging to the functional class of 'signalling and communication' had a positive association with repeats in contrast to the genes belonging to the 'information' class that were negatively associated with repeats.

Background and Objectives: Maintenance of pulpal health is a critical prerequisite for successful application of light amplification by stimulated emission of radiations (lasers) in the hard tissue management of vital teeth. The purpose of this study was to investigate the short- and long-term pulpal effects to cavity-preparations in healthy human teeth using erbium-doped:yttrium, aluminum, and garnet (Er:YAG) laser., Materials and Methods: A total of seven healthy third molars that were to be removed due to space-problem were used. Following the laser excavation, the cavities in dentine were closed temporarily and the teeth were extracted after 7 days (n = 5) and 3 months (n = 2) post-operation. The specimens were fixed, decalcified, subdivided, and processed for light and transmission electron microscopy., Results: In the short-term group, four of the five laser-drilled teeth did not reveal any pathological changes in the pulp-dentine complex. One tooth showed mild disruption of odontoblasts (OB) and vascular dilatation subjacent to the deepest point of the cavity-preparation with a remaining dentine thickness (RDT) of less than 80 microm. The two teeth under long-term observation revealed distinct apposition of tertiary dentine (TD), lined predominantly with cuboidal cells on its pulpal aspect., Conclusions: These results would allow a conclusion to be drawn that the Er:YAG laser under investigation is a pulp preserving hard-tissue drilling tool when used with the specific energy settings and emitting radiation at a wavelength of 2.94 microm., (Copyright 2003 Wiley-Liss, Inc.)

Objective: This report describes 3 cases of ciliated epithelium-lined radicular cysts among 256 apical periodontitis lesions and also illustrates the occurrence of an Actinomyces-infected periapical cyst., Study Design: Serial and step serial sections of 256 plastic-embedded root apices with attached apical periodontitis lesions that were prepared for a previous investigation were reviewed for the presence of ciliated epithelium-lined radicular cysts. The lesions that were found to have such epithelial lining were examined in a transmission electron microscope to elaborate the fine structure of the ciliated cells., Results: A total of 3 ciliated columnar epithelium-lined cysts was found among the 256 apical periodontitis lesions examined. Two of the lesions also contained stratified squamous epithelium. All 3 lesions affected maxillary premolars. One of the lesions was a true cyst, and the other 2 were periapical pocket cysts. The lumen of 1 of the latter revealed the presence of typical "ray-fungus" actinomycotic colonies., Conclusion: Although the stratified squamous component of the epithelia that lined the radicular cysts reported here may be derived from the cell rests of Malassez, the ciliated epithelial cells may be of sinus origin. Microbial agents from diseased root canals can advance into radicular cysts, particularly in pocket cysts, with the possible threat of such infection in upper posterior teeth spreading into the maxillary sinus.

A series of fluorophore-labeled S-nitrosothiols were synthesized, and their fluorescence enhancements upon removal of the nitroso (NO) group were evaluated either by transnitrosation or by photolysis. It was shown that, with a suitable alkyl linker, the fluorescence intensity of dansyl-labeled S-nitrosothiols could be enhanced up to 30-fold. The observed fluorescence enhancement was attributed to the intramolecular energy transfer from fluorophore to the SNO moiety. Ab initio density functional theory (DFT) calculations indicated that the "overlap" between the SNO moiety and the dansyl ring is favored because of their stabilizing interaction, which was in turn affected by both the length of the alkyl linker and the rigidity of the sulfonamide unit. In addition, one of the dansyl-labeled S-nitrosothiols was used to explore the kinetics of S-nitrosothiol/thiol transnitrosation and was evaluated as a fluorescence probe of S-nitrosothiol-bound NO transfer in human umbilical vein endothelial cells.

N-dansylhomocysteine (DnsHCys) is quenched on S-nitrosation. The product of this reaction, N-dansyl-S-nitrosohomocysteine, is a sensitive, direct fluorogenic substrate for the denitrosation activity of protein disulfide isomerase (PDI) with an apparent K(M) of 2 microM. S-nitroso-BSA (BSA-NO) competitively inhibited this reaction with an apparent K(I) of 1 microM. The oxidized form of DnsHCys, N,N-didansylhomocystine, rapidly accumulated in cells and was reduced to DnsHCys. The fluorescence of DnsHCys-preloaded human umbilical endothelial cells and hamster lung fibroblasts were monitored as a function of extracellular BSA-NO concentration via dynamic fluorescence microscopy. The observed quenching of the DnsHCys fluorescence was an indirect measure of cell surface PDI (csPDI) catalyzed denitrosation of extracellular S-nitrosothiols as decrease or increase in the csPDI levels in HT1080 fibrosarcoma cells correlated with the rate of quenching and the PDI inhibitors, 5,5'-dithio-bis-3-nitrobenzoate and 4-(N-(S-glutathionylacetyl) amino)phenylarsenoxide inhibited quenching. The apparent K(M) values for denitrosation of BSA-NO by csPDI ranged from 12 microM to 30 microM. Depletion of membrane N(2)O(3) with the lipophylic antioxidant, vitamin E, inhibited csPDI-mediated quenching rates of DnsHCys fluorescence by approximately 70%. The K(M) for BSA-NO increased by approximately 3-fold and V(max) decreased by approximately 4-fold. These findings suggest that csPDI catalyzed NO released from extracellular S-nitrosothiols accumulates in the membrane where it reacts with O2 to produce N(2)O(3). Intracellular thiols may then be nitrosated by N2O3 at the membrane-cytosol interface.