Respiratory syncytial virus infection influences tight junction integrity.

Respiratory syncytial virus (RSV) is an important risk factor of asthma development and is responsible for severe respiratory tract infections. However, the influence of RSV infection on barrier function of bronchial epithelial cells in vitro and in vivo is still unclear. The aim of this study was to analyse the role of RSV in tight junction (TJ) regulation and to compare epithelial integrity between asthmatic and healthy individuals upon RSV infection. Healthy and asthmatic human bronchial epithelial cells (HBECs) were differentiated at air-liquid interface (ALI) and infected with RSV and ultraviolet (UV)-irradiated RSV. TJ expression and their integrity were analysed by quantitative polymerase chain reaction (qPCR), transepithelial resistance (TER) and paracellular flux. To determine the effect in vivo, BALB/c mice were infected intranasally with RSV or UV-irradiated RSV A2. Bronchoalveolar lavage and TJ integrity were analysed on days 1, 2, 4 and 6 post-infection by qPCR, bioplex and confocal microscopy. RSV increased barrier integrity in ALI cultures of HBEC from healthy subjects, but no effect was found in HBECs from asthmatics. This was not associated with an increase in TJ mRNA expression. In vivo, RSV induced lung inflammation in mice and down-regulated claudin-1 and occludin mRNA expression in whole lungs. Surprisingly, RSV infection was not observed in bronchial epithelial cells, but was found in the lung parenchyma. Decreased expression of occludin upon RSV infection was visible in mouse bronchial epithelial cells in confocal microscopy. However, there was no regulation of claudin-1 and claudin-7 at protein level.

Biotin Switch Assays for Quantitation of Reversible Cysteine Oxidation.

Thiol groups in protein cysteine residues can be subjected to different oxidative modifications by reactive oxygen/nitrogen species. Reversible cysteine oxidation, including S-nitrosylation, S-sulfenylation, S-glutathionylation, and disulfide formation, modulate multiple biological functions, such as enzyme catalysis, antioxidant, and other signaling pathways. However, the biological relevance of reversible cysteine oxidation is typically underestimated, in part due to the low abundance and high reactivity of some of these modifications, and the lack of methods to enrich and quantify them. To facilitate future research efforts, this chapter describes detailed procedures to target the different modifications using mass spectrometry-based biotin switch assays. By switching the modification of interest to a biotin moiety, these assays leverage the high affinity between biotin and avidin to enrich the modification. The use of stable isotope labeling and a range of selective reducing agents facilitate the quantitation of individual as well as total reversible cysteine oxidation. The biotin switch assay has been widely applied to the quantitative analysis of S-nitrosylation in different disease models and is now also emerging as a valuable research tool for other oxidative cysteine modifications, highlighting its relevance as a versatile, robust strategy for carrying out in-depth studies in redox proteomics.

The Human Proteome Organization Chromosome 6 Consortium: integrating chromosome-centric and biology/disease driven strategies.

The Human Proteome Project (HPP) is designed to generate a comprehensive map of the protein-based molecular architecture of the human body, to provide a resource to help elucidate biological and molecular function, and to advance diagnosis and treatment of diseases. Within this framework, the chromosome-based HPP (C-HPP) has allocated responsibility for mapping individual chromosomes by country or region, while the biology/disease HPP (B/D-HPP) coordinates these teams in cross-functional disease-based groups. Chromosome 6 (Ch6) provides an excellent model for integration of these two tasks. This metacentric chromosome has a complement of 1002-1034 genes that code for known, novel or putative proteins. Ch6 is functionally associated with more than 120 major human diseases, many with high population prevalence, devastating clinical impact and profound societal consequences. The unique combination of genomic, proteomic, metabolomic, phenomic and health services data being drawn together within the Ch6 program has enormous potential to advance personalized medicine by promoting robust biomarkers, subunit vaccines and new drug targets. The strong liaison between the clinical and laboratory teams, and the structured framework for technology transfer and health policy decisions within Canada will increase the speed and efficacy of this transition, and the value of this translational research. BIOLOGICAL SIGNIFICANCE: Canada has been selected to play a leading role in the international Human Proteome Project, the global counterpart of the Human Genome Project designed to understand the structure and function of the human proteome in health and disease. Canada will lead an international team focusing on chromosome 6, which is functionally associated with more than 120 major human diseases, including immune and inflammatory disorders affecting the brain, skeletal system, heart and blood vessels, lungs, kidney, liver, gastrointestinal tract and endocrine system. Many of these chronic and persistent diseases have a high population prevalence, devastating clinical impact and profound societal consequences. As a result, they impose a multi-billion dollar economic burden on Canada and on all advanced societies through direct costs of patient care, the loss of health and productivity, and extensive caregiver burden. There is no definitive treatment at the present time for any of these disorders. The manuscript outlines the research which will involve a systematic assessment of all chromosome 6 genes, development of a knowledge base, and development of assays and reagents for all chromosome 6 proteins. We feel that the informatic infrastructure and MRM assays developed will place the chromosome 6 consortium in an excellent position to be a leading player in this major international research initiative. This article is part of a Special Issue: Can Proteomics Fill the Gap Between Genomics and Phenotypes?

Parkin is transcriptionally regulated by ATF4: evidence for an interconnection between mitochondrial stress and ER stress.

Loss of parkin function is responsible for the majority of autosomal recessive parkinsonism. Here, we show that parkin is not only a stress-protective, but also a stress-inducible protein. Both mitochondrial and endoplasmic reticulum (ER) stress induce an increase in parkin-specific mRNA and protein levels. The stress-induced upregulation of parkin is mediated by ATF4, a transcription factor of the unfolded protein response (UPR) that binds to a specific CREB/ATF site within the parkin promoter. Interestingly, c-Jun can bind to the same site, but acts as a transcriptional repressor of parkin gene expression. We also present evidence that mitochondrial damage can induce ER stress, leading to the activation of the UPR, and thereby to an upregulation of parkin expression. Vice versa, ER stress results in mitochondrial damage, which can be prevented by parkin. Notably, the activity of parkin to protect cells from stress-induced cell death is independent of the proteasome, indicating that proteasomal degradation of parkin substrates cannot explain the cytoprotective activity of parkin. Our study supports the notion that parkin has a role in the interorganellar crosstalk between the ER and mitochondria to promote cell survival under stress, suggesting that both ER and mitochondrial stress can contribute to the pathogenesis of Parkinson's disease.

Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity.

The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.

Molecular characterization of a tetramolecular complex between dsDNA and a DNA-binding leucine zipper peptide dimer by mass spectrometry.

The characterization of sequence-specific noncovalent complexes of the GCN4 peptides and dsDNA using mass spectrometry is reported. The GCN4 peptides belong to a class of proteins which bind to sequence-specific dsDNA and are important in the regulation of gene transcription in yeast. These proteins contain a bZIP structural motif which consists of a basic DNA-binding domain and a leucine zipper dimerization domain. The protein dimers specifically bind double-stranded DNA containing the binding element 5'-ATGA(C/G)TCAT-3' to form a tetramolecular noncovalent complex. Using electrospray ionization, we report the detection of such a specific tetramolecular complex using mass spectrometry. Under conditions necessary for observation of the tetramolecular complex, no ions were detected for the GCN4 peptide dimer or the GCN4 monomer with dsDNA. These observations indicate that the specific interaction of the dsDNA with the protein dimer stabilizes the biologically significant noncovalent complex in the gas phase. Complexes were observed for various lengths of both blunt-ended and cohesive-ended double-stranded DNA containing the specific recognition sequence. The binding specificity of the complex was verified with the use of control DNA not containing the recognition sequence and control peptides not known to bind DNA specifically. Additionally, combining limited proteolysis of GCN4 peptide-DNA complexes with mass spectrometric determination of the products compared to identical experiments with noncomplexed peptides was used to probe interactions of specific amino acids with the DNA. The ability to observe these complexes by mass spectrometry and to probe the specific interactions involved opens the door for utilizing this analytical technique to other structural biological problems including the study of transcription processes and determining the specific binding regions between dsDNA and proteins.

Direct monitoring of protein-chemical reactions utilising nanoelectrospray mass spectrometry.

The feasibility of nanoelectrospray mass spectrometry (nanoESI) for the direct analysis of protein chemical reactions and structural changes of proteins has been evaluated. Taking advantage of the long spraying time and the capability of nanoESI for employing a wide range of solvent conditions such as buffers and detergents, applications of monitoring reaction pathways, and dynamics have been carried out with several peptides and proteins. The time course of proteolytic digestions with trypsin and pepsin was investigated for several model polypeptides, and nanoESI showed to provide an efficient tool for optimising digestion conditions for the mass spectrometric peptide mapping analysis. Examples of specific protein chemical modification reactions at arginine and tyrosine residues illustrate the feasibility of nanoESI to monitoring reaction yields and modification sites for more than 180 min. Furthermore, changes of the pattern of protonated molecules caused by temperature effects and by protein unfolding due to disulfide bond reduction have been studied with the model proteins cytochrome c and hen eggwhite lysozyme. The results indicate that nanoESI is an efficient technique for the direct, molecular characterisation of protein-chemical reactions in solution.

The enigma of lambdoid positional molding.

One hundred and twenty-one consecutive patients presenting to the Children's National Medical Center with lambdoid positional molding (LPM) were reviewed. Clinical features included unilateral occipital flattening and alopecia and forward displacement of the ipsilateral ear, forehead, and maxilla. Head tilt and tightness of the ipsilateral sternocleidomastoid muscle were common. An unexplained preponderance of LPM was found in males (74%) and on the right side (72%), both findings statistically significant (p < 0.001). Importantly, a variety of other abnormalities were seen with LPM: torticollis (41%), large head circumference (40), excess extra-axial cerebrospinal fluid (35), developmental delay (19), and other CNS abnormalities (20%). Systemic problems affecting the mobility were also common. Only 3 patients had craniosynostosis, and only 2 with LPM required surgery for severe cosmetic deformities. An apparent increase in the incidence of LPM was attributed to current recommendations to keep infants supine to decrease the risk of sudden infant death syndrome, overutilization of infant carriers similar to cradleboards of earlier cultures, and neonatal medical problems resulting in relative immobility. No evidence was found to support the concept that LPM causes compressive brain pathology; thus, surgical treatment is not required for such fears. Further, the sequelae of underlying CNS and systemic problems associated with LPM would not be corrected by opening unfused sutures, but could even be misinterpreted as complications of surgery.

Characterization of specific noncovalent protein complexes by UV matrix-assisted laser desorption ionization mass spectrometry.

While electrospray (ESI) mass spectrometry has already established its potential for the characterization of non-covalent protein complexes, matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) seemed not to be applicable hitherto because of limitations in matrix chemistry and sample preparation. In this work, a sample preparation method has been developed in which 6-aza-2-thiothymine (ATT) was used as a matrix without any addition of organic cosolvents, and proteins were dissolved in aqueous buffers such as ammonium hydrogencarbonate, ammonium citrate and ammonium acetate. Under these conditions, the intact non-covalent protein complexes, RNAse S, the non-covalent complex of S-protein and S-peptide and specific dimers of coiled-coil leucine zipper polypeptides were observed by UV-MALDI/MS. The specificity of complex formation was ascertained by admixture of non-specific peptides which did not yield detectable aggregate ions. In addition, on-target tryptic digestion of cytochrome c and leucine zipper peptides was carried out after MALDI/MS molecular mass determination in the presence of the ATT matrix. Mass spectrometric analyses of these tryptic digests yielded spectra that showed complete digestion of the proteins. These results indicate that proteins maintained intact tertiary structures necessary for the formation of specific non-covalent complexes, and that trypsin retained its functional enzymatic structure and full biological activity with the present sample preparation method.

Mass spectrometric approaches to molecular characterization of protein-nucleic acid interactions.

The recent development of 'soft' ionization-desorption methods has lead to a breakthrough for the mass spectrometric analysis of biomacromolecules such as proteins and nucleic acids. In particular, the feasibility of electrospray-ionization mass spectrometry (ESI-MS) for the direct characterization of non-covalent supramolecular complexes is opening new analytical perspectives. Examples hitherto analyzed by ESI-MS include enzyme-substrate and -inhibitor complexes, homo- and heterodimers/trimers of leucine zipper polypeptides, and several other DNA- and RNA-binding proteins. Furthermore, the characterization of double-stranded and higher-order oligo- and polynucleotide complexes by negative-ion ESI has been demonstrated. Ions specific of non-covalent protein and oligonucleotide complexes can be selectively dissociated by changing the solution conditions and by increasing the desolvation potential. These results form the basis for the molecular characterization of protein-nucleotide interactions, thus complementing protein-chemical approaches, and other methods of structure determination.

Pediatric cerebellar hemorrhages.

Cerebellar hemorrhage is a devastating condition with morbidity and mortality related not only to the etiology of the hemorrhage, but also to the timing of the intervention. Sixteen consecutive pediatric patients with acute cerebellar hemorrhages are presented: 6 had vascular abnormalities, 3 had tumors, and 2 had hemorrhages of unknown etiology. Thirteen of the 16 patients survived with only 1 of the 13 having persistent vegetative state as a neurologic outcome. Six of 8 patients presenting in a moribund condition had good outcomes, and 3 of 4 patients presenting with fixed and dilated pupils also had good outcomes. Thus, in contrast to adults, rapid evaluation by CT scanning, followed by the judicious use of ventricular drainage and prompt surgical treatment, have resulted in favorable outcomes in pediatric patients despite their poor clinical presentations. None of the neonates having cerebellar hemorrhages required surgical intervention; their courses could be followed clinically and with transfontanel ultrasound.

Time-related patterns of ventricular shunt failure.

Proximal obstruction is reported to be the most common cause of ventriculoperitoneal (VP) shunt failure, suggesting that imperfect ventricular catheter placement and inadequate valve mechanisms are major causes. This study retrospectively examined patterns of shunt failure in 128 consecutive patients with symptoms of shunt malfunction over a 2-year period. Factors analyzed included site of failure, time from shunt placement or last revision to failure, age of patient at time of failure, infections, and primary etiology of the hydrocephalus. One hundred of these patients required revisions; 14 revisions were due to infections. In this series there was a higher incidence of distal (43%) than of proximal (35%) failure. The difference was not statistically significant when the overall series was considered; however, when factoring time to failure as a variable, marked differences were noted regardless of the underlying cause of hydrocephalus or the age of the patient. Of the 49 patients needing a shunt revision or replacement within 2 years of the previous operation, 50% had proximal malfunction, 14% distal, and 10% had malfunctions attributable directly to the valve itself. Also, 12 of the 14 infections occurred during this time interval. In sharp contrast, of the 51 patients having shunt failure from 2 to more than 12 years after the previous procedure, 72% had distal malfunction, 21% proximal, and only 6% had a faulty valve or infection. This difference between time to failure for proximal versus distal failures was statistically significant (P < 0.00001 for both Student's t-test and non-parametric Mann-Whitney U-test).(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphate catalyzed exchange rates of NH-N protons of DNA.

We have studied the catalysis of the exchange of the hydrogen-bonded NH-N protons of the short DNA helix (d-CCAAGCTTGG)2 by phosphate addition. The NH exchange rates were monitored by the line widths of the corresponding NH resonances in the 1H nmr spectra. The exchange catalyst phosphate is most effective on the exchange rate of the terminal CG1 base pairs. However, all internal base pairs are also moderately affected by phosphate which suggests an exchange mechanism governed by a fast equilibrium between opened and closed states of the duplex. Within the limits of error the same effectiveness of phosphate on the exchange rate of all internal NH-N protons has been observed. With the exception of the terminal base pairs, no sequence and/or position specificity of the exchange rates of the NH-N protons of the base pairs has been found.

Computer programming for nucleic acid studies. II. Total chemical shifts calculation of all protons of double-stranded helices.

A FORTRAN computer program called SHIFTS is described. Through SHIFTS, one can calculate the NMR chemical shifts of the proton resonances of single and double-stranded nucleic acids of known sequences and of predetermined conformations. The program can handle RNA and DNA for an arbitrary sequence of a set of 4 out of the 6 base types A,U,G,C,I and T. Data files for the geometrical parameters are available for A-, A'-, B-, D- and S-conformations. The positions of all the atoms are calculated using a modified version of the SEQ program [1]. Then, based on this defined geometry three chemical shift effects exerted by the atoms of the neighboring nucleotides on the protons of each monomeric unit are calculated separately: the ring current shielding effect: the local atomic magnetic susceptibility effect (including both diamagnetic and paramagnetic terms); and the polarization or electric field effect. Results of the program are compared with experimental results for a gamma (ApApGpCpUpU) 2 helical duplex and with calculated results on this same helix based on model building of A'-form and B-form and on graphical procedure for evaluating the ring current effects.

Computer programs for nucleic acids studies. Atomic coordinates of helices and their graphic display.

For the purpose of calculation of NMR and other physiocochemical properties of nucleic acids, a computer program in FORTRAN language has been written. This program provides the printout of the Cartesian and cylindrical coordinates of all atoms of a double-stranded helix of nucleic acid in either A, A' or B conformation with any specified base sequence up to 50 nucleotides or longer. In addition, the interatomic distances between any two atoms or distances (with both perpendicular and parallel components) from the centers of the base rings to any atom in the helix can be calculated. This information has been used for the calculation of the ring current effects of the 1H chemical shift of two short helices. Satisfactory agreement has been found in the comparison between the present data and that obtained from model construction and from the table prepared by Arter and Schmidt. The structure of the helix can also be illustrated in graphic form on a Tektronix 4006 CRT terminal. The presentation can be manipulated, such as selection, enlargement, translation and rotation.

Scanning kirkpatrick-baez x-ray telescope to maximize effective area and eliminate spurious images; design.

We consider the design of a Kirkpatrick-Baez grazing-incidence X-ray telescope to be used in a scan of the sky and analyze the distribution of both properly reflected rays and spurious images over the field of view. To obtain maximum effective area over the field of view, it is necessary to increase the spacing between plates for a scanning telescope as compared to a pointing telescope. Spurious images are necessarily present in this type of lens, but they can be eliminated from the field of view by adding properly located baffles or collimators. Results of a computer design are presented.