Proteoforms in Peripheral Blood Mononuclear Cells as Novel Rejection Biomarkers in Liver Transplant Recipients.

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent "top-down" proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight-based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum-rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management.

Genetic variation in blue whales in the eastern pacific: implication for taxonomy and use of common wintering grounds.

Many aspects of blue whale biology are poorly understood. Some of the gaps in our knowledge, such as those regarding their basic taxonomy and seasonal movements, directly affect our ability to monitor and manage blue whale populations. As a step towards filling in some of these gaps, microsatellite and mtDNA sequence analyses were conducted on blue whale samples from the Southern Hemisphere, the eastern tropical Pacific (ETP) and the northeast Pacific. The results indicate that the ETP is differentially used by blue whales from the northern and southern eastern Pacific, with the former showing stronger affinity to the region off Central America known as the Costa Rican Dome, and the latter favouring the waters of Peru and Ecuador. Although the pattern of genetic variation throughout the Southern Hemisphere is compatible with the recently proposed subspecies status of Chilean blue whales, some discrepancies remain between catch lengths and lengths from aerial photography, and not all blue whales in Chilean waters can be assumed to be of this type. Also, the range of the proposed Chilean subspecies, which extends to the Galapagos region of the ETP, at least seasonally, perhaps should include the Costa Rican Dome and the eastern North Pacific as well.

Signaling switch of the urotensin II vasosactive peptide GPCR: prototypic chemotaxic mechanism in glioma.

Multiform glioblastomas (GBM) are the most frequent and aggressive primary brain tumors in adults. The poor prognosis is due to neo-angiogenesis and cellular invasion, processes that require complex chemotaxic mechanisms involving motility, migration and adhesion. Understanding these different cellular events implies identifying receptors and transduction pathways that lead to and promote either migration or adhesion. Here we establish that glioma express the vasoactive peptide urotensin II (UII) and its receptor UT and that UT-mediated signaling cascades are involved in glioma cell migration and adhesion. Components of the urotensinergic systems, UII and UT, are widely expressed in patient-derived GBM tissue sections, glioma cell lines and fresh biopsy explants. Interestingly, gradient concentrations of UII produced chemoattracting migratory/motility effects in glioma as well as HEK293 cells expressing human UT. These effects mainly involved the G13/Rho/rho kinase pathway while partially requiring Gi/o/PI3K components. In contrast, we observed that homogeneous concentrations of UII drastically blocked cell motility and stimulated cell-matrix adhesions through a UT/Gi/o signaling cascade, partially involving phosphatidylinositol-3 kinase. Finally, we provide evidence that, in glioma cells, homogeneous concentration of UII allowed translocation of Gα13 to the UT receptor at the plasma membrane and increased actin stress fibers, lamellipodia formation and vinculin-stained focal adhesions. UII also provoked a re-localization of UT precoupled to Gαi in filipodia and initiated integrin-stained focal points. Altogether, these findings suggest that UT behaves as a chemotaxic receptor, relaying a signaling switch between directional migration and cell adhesion under gradient or homogeneous concentrations, thereby redefining sequential mechanisms affecting tumor cells during glioma invasion. Taken together, our results allow us to propose a model in order to improve the design of compounds that demonstrate signaling bias for therapies that target specifically the Gi/o signaling pathway.

Physiological effects of heat stress on Hawaiian picture-wing Drosophila: genome-wide expression patterns and stress-related traits.

Climate change is compounding the threats to the future of biodiversity, already impacted by habitat loss, invasive species and diseases. In the Hawaiian Islands, many of the endemic species have narrow habitat ranges that make them especially vulnerable to climate change. The Hawaiian Drosophila, a remarkably diverse group of species with 11 listed as federally endangered, are thought to be sensitive to temperature changes. To examine the species differences in sensitivity of Hawaiian picture-wing Drosophila to temperature changes, wild populations of Drosophila sproati, a relatively common species, and Drosophila silvestris, a rare species, were collected from two locations on Hawaii Island and bred in common laboratory conditions. Adult flies were exposed to hot and cold temperatures and compared with adult flies at control temperatures. Drosophila silvestris adults were less tolerant to heat stress than D. sproati for both survival and sperm mobility. In contrast, D. silvestris adults were more tolerant to cold stress than D. sproati for adult survival. The expression of 4950 Gene Ontology annotated gene transcripts was also analysed in high-temperature-treated and control males to identify candidate genes related to heat tolerance. There were more than twice as many transcripts differentially expressed after high temperature treatment for D. silvestris (246 transcripts) as for D. sproati (106 transcripts), with 13 Gene Ontology terms enriched between temperatures for D. silvestris and merely three in D. sproati. The combined results are consistent with D. sproati occurring more widely today as well as occurring at lower elevations than D. silvestris and with a genetically based temperature response, which is more severe in D. silvestris at high temperatures than that in D. sproati. These experiments demonstrate the potential for different capacities of species to adapt to future climate change conditions as well as providing an explanation for historical changes in the distribution of species.

Blue whale population structure along the eastern South Pacific Ocean: evidence of more than one population.

Blue whales (Balaenoptera musculus) were among the most intensively exploited species of whales in the world. As a consequence of this intense exploitation, blue whale sightings off the coast of Chile were uncommon by the end of the 20th century. In 2004, a feeding and nursing ground was reported in southern Chile (SCh). With the aim to investigate the genetic identity and relationship of these Chilean blue whales to those in other Southern Hemisphere areas, 60 biopsy samples were collected from blue whales in SCh between 2003 and 2009. These samples were genotyped at seven microsatellite loci and the mitochondrial control region was sequenced, allowing us to identify 52 individuals. To investigate the genetic identity of this suspected remnant population, we compared these 52 individuals to blue whales from Antarctica (ANT, n = 96), Northern Chile (NCh, n = 19) and the eastern tropical Pacific (ETP, n = 31). No significant differentiation in haplotype frequencies (mtDNA) or among genotypes (nDNA) was found between SCh, NCh and ETP, while significant differences were found between those three areas and Antarctica for both the mitochondrial and microsatellite analyses. Our results suggest at least two breeding population units or subspecies exist, which is also supported by other lines of evidence such as morphometrics and acoustics. The lack of differences detected between SCh/NCh/ETP areas supports the hypothesis that eastern South Pacific blue whales are using the ETP area as a possible breeding area. Considering the small population sizes previously reported for the SCh area, additional conservation measures and monitoring of this population should be developed and prioritized.

Lower calcineurin inhibitor doses in older compared to younger kidney transplant recipients yield similar troughs.

The number of older adults undergoing kidney transplantation has increased, yet little is known about calcineurin inhibitor (CNI) metabolism in this group. We studied CNI troughs and doses to determine if there were age-related differences in metabolism and dose requirements. We studied 348 young (18-34 years), 1831 middle (35-64 years) and 374 older (65-84 years) adult kidney transplant recipients enrolled in a seven-center prospective study. Troughs were obtained from each patient 2×/week in weeks 1-8 and 2×/month in months 3-6. A multivariable linear-mixed model examined the effect of age on log dose and weight normalized troughs. Older recipients had higher normalized tacrolimus troughs than middle or young age adults despite receiving doses a median of 1-2 mg/day lower. Age and CYP3A5*1 genotype had the largest effect on tacrolimus troughs. Older recipients also had higher normalized cyclosporine troughs than middle or young adults despite receiving median doses 100 mg/day lower. After normalization for dose and weight, CNI troughs were more than 50% higher in older adults than young adults. These data support age-related changes in CNI metabolism. Further studies are needed to determine optimal dosing of CNIs in the elderly.

Inflammation in areas of tubular atrophy in kidney allograft biopsies: a potent predictor of allograft failure.

The Banff scoring schema provides a common ground to analyze kidney transplant biopsies. Interstitial inflammation (i) and tubulitis (t) in areas of viable tissue are features in scoring acute rejection, but are excluded in areas of tubular atrophy (TA). We studied inflammation and tubulitis in a cohort of kidney transplant recipients undergoing allograft biopsy for new-onset late graft dysfunction (N = 337). We found inflammation ('iatr') and tubulitis ('tatr') in regions of fibrosis and atrophy to be strongly correlated with each other (p < 0.0001). Moreover, iatr was strongly associated with death-censored graft failure when compared to recipients whose biopsies had no inflammation, even after adjusting for the presence of interstitial fibrosis (Hazard Ratio = 2.31, [1.10-4.83]; p = 0.0262) or TA (hazard ratio = 2.42, [1.16-5.08]; p = 0.191), serum creatinine at the time of biopsy, time to biopsy and i score. Further, these results did not qualitatively change after additional adjustments for C4d staining or donor specific antibody. Stepwise regression identified the most significant markers of graft failure which include iatr score. We propose that a more global assessment of inflammation in kidney allograft biopsies to include inflammation in atrophic areas may provide better prognostic information. Phenotypic characterization of these inflammatory cells and appropriate treatment may ameliorate late allograft failure.

Histopathologic clusters differentiate subgroups within the nonspecific diagnoses of CAN or CR: preliminary data from the DeKAF study.

The nonspecific diagnoses 'chronic rejection''CAN', or 'IF/TA' suggest neither identifiable pathophysiologic mechanisms nor possible treatments. As a first step to developing a more useful taxonomy for causes of new-onset late kidney allograft dysfunction, we used cluster analysis of individual Banff score components to define subgroups. In this multicenter study, eligibility included being transplanted prior to October 1, 2005, having a 'baseline' serum creatinine < or =2.0 mg/dL before January 1, 2006, and subsequently developing deterioration of graft function leading to a biopsy. Mean time from transplant to biopsy was 7.5 +/- 6.1 years. Of the 265 biopsies (all with blinded central pathology interpretation), 240 grouped into six large (n > 13) clusters. There were no major differences between clusters in recipient demographics. The actuarial postbiopsy graft survival varied by cluster (p = 0.002). CAN and CNI toxicity were common diagnoses in each cluster (and did not differentiate clusters). Similarly, C4d and presence of donor specific antibody were frequently observed across clusters. We conclude that for recipients with new-onset late graft dysfunction, cluster analysis of Banff scores distinguishes meaningful subgroups with differing outcomes.

Pathological and clinical characterization of the 'troubled transplant': data from the DeKAF study.

We are studying two cohorts of kidney transplant recipients, with the goal of defining specific clinicopathologic entities that cause late graft dysfunction: (1) prevalent patients with new onset late graft dysfunction (cross-sectional cohort); and (2) newly transplanted patients (prospective cohort). For the cross-sectional cohort (n = 440), mean time from transplant to biopsy was 7.5 +/- 6.1 years. Local pathology diagnoses included CAN (48%), CNI toxicity (30%), and perhaps surprisingly, acute rejection (cellular- or Ab-mediated) (23%). Actuarial rate of death-censored graft loss at 1 year postbiopsy was 17.7%; at 2 years, 29.8%. There was no difference in postbiopsy graft survival for recipients with versus without CAN (p = 0.9). Prospective cohort patients (n = 2427) developing graft dysfunction >3 months posttransplant undergo 'index' biopsy. The rate of index biopsy was 8.8% between 3 and 12 months, and 18.2% by 2 years. Mean time from transplant to index biopsy was 1.0 +/- 0.6 years. Local pathology diagnoses included CAN (27%), and acute rejection (39%). Intervention to halt late graft deterioration cannot be developed in the absence of meaningful diagnostic entities. We found CAN in late posttransplant biopsies to be of no prognostic value. The DeKAF study will provide broadly applicable diagnostic information to serve as the basis for future trials.

10mM glucosamine prevents activation of proADAMTS5 (aggrecanase-2) in transfected cells by interference with post-translational modification of furin.

OBJECTIVE: Glucosamine has been previously shown to suppress cartilage aggrecan catabolism in explant cultures. We determined the effect of glucosamine on ADAMTS5 (a disintegrin-like and metalloprotease domain (reprolysin type) with thrombospondin type-1 motifs 5), a major aggrecanase in osteoarthritis, and investigated a potential mechanism underlying the observed effects. DESIGN: HEK293F and CHO-K1 cells transiently transfected with ADAMTS5 cDNA were treated with glucosamine or the related hexosamine mannosamine. Glucosamine effects on FURIN transcription were determined by quantitative RT-PCR. Effects on furin-mediated processing of ADAMTS5 zymogen, and aggrecan processing by glucosamine-treated cells, were determined by western blotting. Post-translational modification of furin and N-glycan deficient furin mutants generated by site-directed mutagenesis was analyzed by western blotting, and the mutants were evaluated for their ADAMTS5 processing ability in furin-deficient CHO-RPE.40 cells. RESULTS: Ten mM glucosamine and 5-10mM mannosamine reduced excision of the ADAMTS5 propeptide, indicating interference with the propeptide excision mechanism, although mannosamine compromised cell viability at these doses. Although glucosamine had no effect on furin mRNA levels, western blot of furin from glucosamine-treated cells suggested altered post-translational modification. Glucosamine treatment led to decreased glycosylation of cellular furin, with reduced furin autoactivation as the consequence. Recombinant furin treated with peptide N-glycanase F had reduced activity against a synthetic peptide substrate. Indeed, site-directed mutagenesis of two furin N-glycosylation sites, Asn(387) and Asn(440), abrogated furin activation and this mutant was unable to rescue ADAMTS5 processing in furin-deficient cells. CONCLUSIONS: Ten mM glucosamine reduces excision of the ADAMTS5 propeptide via interference with post-translational modification of furin and leads to reduced aggrecanase activity of ADAMTS5.

Use of cardioprotective medications in kidney transplant recipients.

Death with function causes half of late kidney transplant failures, and cardiovascular disease (CVD) is the most common cause of death in these patients. We examined the use of potentially cardioprotective medications in a prospective observational study at seven transplant centers in the United States and Canada. Among 935 patients, 87% received antihypertensive medications at both 1 and 6 months after transplantation. Similar antihypertensive regimens were used for patients with and without diabetes and CVD, but with wide variability among centers. In contrast, while 44% of patients were on angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) at the time of transplantation, the proportion taking these agents dropped to 12% at month 1, then increased to 24% at 6 months. Fewer than 30% with CVD or diabetes received ACEI/ARB therapy 6 months posttransplant. Aspirin use was uncommon (<40% of patients). Even among those with diabetes and/or CVD, fewer than 60% received aspirin and only half received a statin at 1 and 6 months. This study demonstrates marked variability in the use of cardioprotective medications in kidney transplant recipients, a finding that may reflect, among several possible explanations, clinical uncertainty due the lack of randomized trials for these medications in this population.

Temperature-dependent variations of ligand-receptor contact points in hAT(1).

Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT(1) has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT(1) receptor and two representative Met mutants (H256M-hAT(1) and F293M-hAT(1)) from the iterative mutagenesis study were photolabelled with the benzophenone-ligand (125)I-[Sar(1), Bpa(8)]AngII at temperatures ranging from - 15 degrees C to 37 degrees C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT(1) and F293M-hAT(1) across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT(1) was observed. At - 15 degrees C, H256M labelling was identical to that of hAT(1), indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37 degrees C labelling occurred almost exclusively at mutated residue H256M-hAT(1) in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures.

Effect of baffles on nitrification in aerated facultative lagoons in a cold climate.

Tracer studies performed in two aerated facultative lagoons indicate some bypass and an overall hydraulic regime close to completely-mixed. Results were used to calibrate a hydraulic model based on the tanks-in-series approach. The hydraulic model was combined with a simple "death-regeneration" biokinetic model to simulate seasonal nitrification as observed over a three year period. Modifications were made to the hydraulic model to represent the effect of baffle installations to 1) eliminate bypass and 2) impose a plug-flow regime. Simulation results indicate there is some gain to eliminating bypass but that imposing a plug-flow regime would increase biomass washout rates and hinder nitrification.

Angiotensin II is bound to both receptors AT1 and AT2, parallel to the transmembrane domains and in an extended form.

UNLABELLED: We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. CONCLUSIONS: (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.

The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin.

A series of mutants incorporating furin recognition sequences within the P6-P1 region of the reactive site loop of alpha(1)-antitrypsin were constructed. Variants containing different combinations of basic residues in the P1, P2, P4, and P6 positions replacing the wild type (P6)LEAIPM(P1) sequence were evaluated for their capacity to establish SDS-resistant complexes with furin, to affect association rate constants (k(ass) and k'(ass)), or to inhibit furin-dependent proteolysis of a model precursor in vivo. Each variant abolished processing of pro-von Willebrand factor in transfected hEK293 cells. The k(ass) of all variants were found to be similar (1.1-1.7 x 10(6) m(-1) s(-1)) except for one mutant, RERIRR, which had a k(ass) of 3.3 x 10(5) m(-1) s(-1). However, the stoichiometry of inhibition varied with values ranging from 2.9 to >24, indicating rapid formation of the acyl-enzyme intermediate (high k'(ass)). Moreover, those variants having high stoichiometry of inhibition values were accompanied by the rapid formation of cleaved forms of the inhibitors. The data suggest that the rate of conversion of the acyl-enzyme (EI') into the highly stable complex (EI*) was affected by replacement of specific residues within the reactive site loop. Taken together, the results reveal how furin recognition sequences within the context of the biochemical properties of serpins will play a role in the capacity of the protein to follow either the inhibitory or the substrate pathway.

Ligand-induced signaling in the absence of furin processing of Notch1.

Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor which is both present on the cell surface and able to generate an intracellular signal in response to ligand. Consistent with previous reports, we found that disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbed ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL (CBF1, Su(H), and LAG-1). Yet contrary to these reports, we could detect the full-length p300 Notch1 product on the cell surface. Moreover, this uncleaved form of Notch1 could suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support our previous studies characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. Our results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor could mediate two distinct signaling pathways in response to ligand.

Evidence that furin is an authentic transforming growth factor-beta1-converting enzyme.

Transforming growth factor (TGF)-beta1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-beta1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-beta1 convertase. Even though six members of the PC family have the ability to cleave TGF-beta1, ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-beta1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-beta1. Moreover, both furin and TGF-beta are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-beta1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-beta system and should facilitate the development of therapeutically useful TGF-beta inhibitors.

Assays for radiolabel-photoaffinity labeling of Angiotensin receptors.

Photoaffinity labeling is a useful method to covalently bind two interacting moieties whether they be substrate and enzyme or ligand and receptor. Irreversibly labeling any particular molecule is a practical way of detecting the latter throughout the course of a characterization or a purification procedure.