Isotope-coded, iodoacetamide-based reagent to determine individual cysteine pK(a) values by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cysteine reactivity in enzymes is imparted to a large extent by the stabilization of the deprotonated form of the reduced cysteine (i.e., the thiolate) within the active site. Although this is likely to be an important chemical attribute of many thiol-based enzymes, including cysteine-dependent peroxidases (peroxiredoxins) and proteases, only relatively few pK(a) values have been determined experimentally. Presented here is a new technique for determining the pK(a) value of cysteine residues through quantitative mass spectrometry following chemical modification with an iodoacetamide-based reagent over a range of pH buffers. This isotope-coded reagent, N-phenyl iodoacetamide (iodoacetanilide), is readily prepared in deuterated (d(5)) and protiated (d(0)) versions and is more reactive toward free cysteine than is iodoacetamide. Using this approach, the pK(a) values for the two cysteine residues in Escherichia coli thioredoxin were determined to be 6.5 and greater than 10.0, in good agreement with previous reports using chemical modification approaches. This technique allows the pK(a) of specific cysteine residues to be determined in a clear, fast, and simple manner and, because cysteine residues on separate tryptic peptides are measured separately, is not complicated by the presence of multiple cysteines within the protein of interest.     

Whole genome amplification on poly(dimethylsiloxane) microchip array

A new method for on-plate protein digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry analysis is proposed involving an automated one-step sample separation using nanoflow HPLC followed by nanoliter fraction collection and on-plate digestion with trypsin. This procedure uses a commercial automatic nanoliter fraction collection system for on-line spotting of the eluent onto a MALDI target. After protein digestion, the reaction is stopped by the addition of acidified matrix using the same automated system. Collected spots are subsequently analyzed using a MALDI tandem time-of-flight (TOF/TOF) mass spectrometer for protein sequencing and identification.     

Mechanisms of human papillomavirus type 16 neutralization by l2 cross-neutralizing and l1 type-specific antibodies

Pseudovirions of human papillomavirus type 16 (HPV16), the principal etiologic agent in 50% of cervical cancers, were used as a model system to investigate the cell surface interactions involved in the exposure of the broadly cross-neutralizing papillomavirus L2 epitopes. These neutralizing epitopes were exposed only after cell surface binding and a subsequent change in capsid conformation that permitted cleavage by the cellular protease furin at a specific highly conserved site in L2 that is immediately upstream of the cross-neutralizing epitopes. Unexpectedly, binding of L2 antibodies led to the release of the capsid/antibody complexes from the cell surface and their accumulation on the extracellular matrix. Study of the dynamics of exposure of the L2 epitopes further revealed that representatives of the apparently dominant class of L1-specific neutralizing antibodies induced by virus-like particle vaccination prevent infection, not by preventing cell surface binding but rather by preventing the conformation change involved in exposure of the L2 neutralizing epitope. These findings suggest a dynamic model of virion-cell surface interactions that has implications for both evolution of viral serotypes and the efficacy of current and future HPV vaccines.     

Substrate cleavage analysis of furin and related proprotein convertases. A comparative study

We present the data and the technology, a combination of which allows us to determine the identity of proprotein convertases (PCs) related to the processing of specific protein targets including viral and bacterial pathogens. Our results, which support and extend the data of other laboratories, are required for the design of effective inhibitors of PCs because, in general, an inhibitor design starts with a specific substrate. Seven proteinases of the human PC family cleave the multibasic motifs R-X-(R/K/X)-R downward arrow and, as a result, transform proproteins, including those from pathogens, into biologically active proteins and peptides. The precise cleavage preferences of PCs have not been known in sufficient detail; hence we were unable to determine the relative importance of the individual PCs in infectious diseases, thus making the design of specific inhibitors exceedingly difficult. To determine the cleavage preferences of PCs in more detail, we evaluated the relative efficiency of furin, PC2, PC4, PC5/6, PC7, and PACE4 in cleaving over 100 decapeptide sequences representing the R-X-(R/K/X)-R downward arrow motifs of human, bacterial, and viral proteins. Our computer analysis of the data and the follow-on cleavage analysis of the selected full-length proteins corroborated our initial results thus allowing us to determine the cleavage preferences of the PCs and to suggest which PCs are promising drug targets in infectious diseases. Our results also suggest that pathogens, including anthrax PA83 and the avian influenza A H5N1 (bird flu) hemagglutinin precursor, evolved to be as sensitive to PC proteolysis as the most sensitive normal human proteins.     

TSG-6 regulates bone remodeling through inhibition of osteoblastogenesis and osteoclast activation

TSG-6 is an inflammation-induced protein that is produced at pathological sites, including arthritic joints. In animal models of arthritis, TSG-6 protects against joint damage; this has been attributed to its inhibitory effects on neutrophil migration and plasmin activity. Here we investigated whether TSG-6 can directly influence bone erosion. Our data reveal that TSG-6 inhibits RANKL-induced osteoclast differentiation/activation from human and murine precursor cells, where elevated dentine erosion by osteoclasts derived from TSG-6(-/-) mice is consistent with the very severe arthritis seen in these animals. However, the long bones from unchallenged TSG-6(-/-) mice were found to have higher trabecular mass than controls, suggesting that in the absence of inflammation TSG-6 has a role in bone homeostasis; we have detected expression of the TSG-6 protein in the bone marrow of unchallenged wild type mice. Furthermore, we have observed that TSG-6 can inhibit bone morphogenetic protein-2 (BMP-2)-mediated osteoblast differentiation. Interaction analysis revealed that TSG-6 binds directly to RANKL and to BMP-2 (as well as other osteogenic BMPs but not BMP-3) via composite surfaces involving its Link and CUB modules. Consistent with this, the full-length protein is required for maximal inhibition of osteoblast differentiation and osteoclast activation, although the isolated Link module retains significant activity in the latter case. We hypothesize that TSG-6 has dual roles in bone remodeling; one protective, where it inhibits RANKL-induced bone erosion in inflammatory diseases such as arthritis, and the other homeostatic, where its interactions with BMP-2 and RANKL help to balance mineralization by osteoblasts and bone resorption by osteoclasts.     

The influence of age on the use of potential enrichment objects and synchronisation of behaviour of pigs

This experiment aimed to identify how a pig's age affected the extent and synchrony of use of different environmental enrichment materials, and how this use changed over time. Ten diverse novel objects were each presented to three replicate litters of 3 weeks of age (sucklers) and three replicate groups of three animals of 5 (weaners) weeks and 13 (growers) weeks of age. Video recordings were made of the pigs’ behaviour over a period of 5 days and subsequently analysed for activity, inactivity and object directed behaviour of three animals per group on days 1 and 5. The observed performance of any given behaviour, when at least one other member of the group was also performing that behaviour, was compared with the probability that such concurrence occurred by chance and these results were used to calculate the degree of synchronisation. Gender had no effect on the duration of object use or approach latency. Growers displayed a shorter latency to approach the objects initially compared to sucklers and weaners (mean seconds; 2153 versus 2660 versus 980 for sucklers, weaners and growers, respectively, S.E.D. 542, p = 0.007). Sucklers used the objects to a much lesser extent than either the weaners or growers (mean duration object interaction (s); 129 versus 1253 versus 1412 for sucklers, weaners and growers, respectively, S.E.D. 93, p < 0.001). Overall object use decreased between days 1 and 5 (mean duration object interaction (s); 1326 versus 536 for days 1 and 5, respectively, S.E.D. 76, p < 0.001). All of the age groups synchronised their behaviour to a much greater extent than expected by chance. The sucklers showed a higher degree of synchrony of activity (K; 0.890 versus 0.776 versus 0.682 for sucklers, weaners and growers respectively, S.E.D. 0.013, p < 0.001) and inactivity (K; 0.963 versus 0.905 versus 0.909 respectively, S.E.D. 0.007, p < 0.001), but lower degree of synchrony for object directed behaviour (K; 0.149 versus 0.375 versus 0.370 respectively, S.E.D. 0.025, p < 0.001), than the weaners and growers. The degree of synchronisation of object directed behaviour decreased over the 5-day period, irrespective of age (K; 0.382 versus 0.214 for days 1 and 5 respectively, S.E.D. 0.020, p < 0.001). Significant correlations (r = 0.678–0.879) were found between the degree of synchrony and extent of object interaction only for the sucklers. Since pigs showed behavioural synchronisation, object availability should be considered when providing desirable enrichment in order to avoid excessive competition in larger commercial groups.     

Objective measures of health and well-being in laboratory rhesus monkeys (Macaca mulatta)

BACKGROUND: Non-human primates are an invaluable part of biomedical research. Strict regulations insure animals have a maximum likelihood of well-being and optimum health during the course of experimental procedures. Objective assessment of well-being is a critical component of these assurances. METHODS: Here we describe an objective and quantitative system we used to identify two well-being concerns in laboratory rhesus monkeys (Macaca mulatta). We provide a series of indicators for use by laboratory personnel to promote laboratory primate well-being. The indicators measure (1) potentially life threatening clinical concerns, (2) developing clinical issues, (3) atypical behaviors, and (4) laboratory performance. We include specific criteria to facilitate veterinary intervention. RESULTS: The assessment, applied to two case studies reported here, enabled swift veterinary intervention returning the animals to a healthy state. CONCLUSIONS: The measures described here provide a battery of observable and objective measures across multiple dimensions that can further ensure both excellent science and veterinary care.