Detection of candidate biomarkers of prostate cancer progression in serum: a depletion-free 3D LC/MS quantitative proteomics pilot study.

BACKGROUND: Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease. METHODS: We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa. RESULTS: We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an 'interactome' with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6. CONCLUSIONS: Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.

Simultaneous measurements of cytosolic calcium and secretion in single bovine adrenal chromaffin cells by fluorescent imaging of fura-2 in cocultured cells.

The cytosolic free calcium concentration ([Ca2+]i) and exocytosis of chromaffin granules were measured simultaneously from single, intact bovine adrenal chromaffin cells using a novel technique involving fluorescent imaging of cocultured cells. Chromaffin cell [Ca2+]i was monitored with fura-2. To simultaneously follow catecholamine secretion, the cells were cocultured with fura-2-loaded NIH-3T3t cells, a cell line chosen because of their irresponsiveness to chromaffin cell secretagogues but their large Ca2+ response to ATP, which is coreleased with catecholamine from the chromaffin cells. In response to the depolarizing stimulus nicotine (a potent secretagogue), chromaffin cell [Ca2+]i increased rapidly. At the peak of the response, [Ca2+]i was evenly distributed throughout the cell. This elevation in [Ca2+]i was followed by a secretory response which originated from the entire surface of the cell. In response to the inositol 1,4,5-trisphosphate (InsP3)-mobilizing agonist angiotensin II (a weak secretagogue), three different responses were observed. Approximately 30% of chromaffin cells showed no rise in [Ca2+]i and did not secrete. About 45% of the cells responded with a large (greater than 200 nM), transient elevation in [Ca2+]i and no detectable secretory response. The rise in [Ca2+]i was nonuniform, such that peak [Ca2+]i was often recorded only in one pole of the cell. And finally, approximately 25% of cells responded with a similar Ca2+-transient to that described above, but also gave a secretory response. In these cases secretion was polarized, being confined to the pole of the cell in which the rise in [Ca2+]i was greatest.(ABSTRACT TRUNCATED AT 250 WORDS)

ACAPs are arf6 GTPase-activating proteins that function in the cell periphery.

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

Cytohesins and centaurins: mediators of PI 3-kinase-regulated Arf signaling.

Receptor-activated phosphoinositide (PI) 3-kinases produce PtdIns(3, 4,5)P(3) and its metabolite PtdIns(3,4)P(2) that function as second messengers in membrane recruitment and activation of target proteins. The cytohesin and centaurin protein families are potential targets for PtdIns(3,4,5)P(3) that also regulate and interact with Arf GTPases. Consequently, these families are poised to transduce PI 3-kinase activation into coordinated control of Arf-dependent pathways. Proposed downstream events in PI 3-kinase-regulated Arf cascades include modulation of vesicular trafficking and the actin cytoskeleton.

GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro.

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

Chondroprotection by urocortin involves blockade of the mechanosensitive ion channel Piezo1.

Osteoarthritis (OA) is characterised by progressive destruction of articular cartilage and chondrocyte cell death. Here, we show the expression of the endogenous peptide urocortin1 (Ucn1) and two receptor subtypes, CRF-R1 and CRF-R2, in primary human articular chondrocytes (AC) and demonstrate its role as an autocrine/paracrine pro-survival factor. This effect could only be removed using the CRF-R1 selective antagonist CP-154526, suggesting Ucn1 acts through CRF-R1 when promoting chondrocyte survival. This cell death was characterised by an increase in p53 expression, and cleavage of caspase 9 and 3. Antagonism of CRF-R1 with CP-154526 caused an accumulation of intracellular calcium (Ca2+) over time and cell death. These effects could be prevented with the non-selective cation channel blocker Gadolinium (Gd3+). Therefore, opening of a non-selective cation channel causes cell death and Ucn1 maintains this channel in a closed conformation. This channel was identified to be the mechanosensitive channel Piezo1. We go on to determine that this channel inhibition by Ucn1 is mediated initially by an increase in cyclic adenosine monophosphate (cAMP) and a subsequent inactivation of phospholipase A2 (PLA2), whose metabolites are known to modulate ion channels. Knowledge of these novel pathways may present opportunities for interventions that could abrogate the progression of OA.

Urocortin--from Parkinson's disease to the skeleton.

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis.

Tumor promoter 12-O-tetradecanoylphorbol 13-acetate inhibits mas/angiotensin receptor-stimulated inositol phosphate production and intracellular Ca2+ elevation in the 401L-C3 neuronal cell line.

Stimulation of mas-oncogene transfected 401L-C3 cells by angiotensins leads to the production of inositol phosphates. This response shows dose dependence, and has an apparent rank order of potency angiotensin III greater than or equal to angiotensin II much greater than angiotensin I. Preincubation with 12-O-tetradecanoylphorbol 13-acetate, for 5 min, significantly diminishes both inositol phosphate and intracellular [Ca2+] responses to angiotensins, without affecting those stimulated by the endogenous bradykinin receptor. Incubation of 401L-C3 cells with either phorbol ester or angiotensins leads to elevation of intracellular pH, implying that mas/angiotensin receptor stimulation itself leads to protein kinase C activation. These results suggest the operation of a negative feedback loop specific for the mas/angiotensin receptor signalling pathway, and which may be essential in defining the final biological output response to this receptor stimulation.

Amygdaloid connections with posterior insular and temporal cortical areas in the rat.

The connections of the amygdala with the insular and temporal cortices were examined by injecting wheat germ agglutinin conjugated to HRP (WGA-HRP) into the rat cortex. Following injections into the posterior agranular insular area (AIp) or perirhinal cortex (PR), bands of labeled neurons extending across nuclear boundaries were observed in the amygdala. These neuronal bands involved cells in the lateral, basolateral, and basomedial nuclei as well as the periamygdaloid cortex. Other nuclei of the corticomedial amygdala and the ventral endopiriform nucleus also exhibited retrogradely labeled cells. Anterograde label was observed in nuclei containing labeled neurons and in the central nucleus. Injections into gustatory, somatosensory, and auditory neocortical areas located dorsal to AIp and PR labeled small numbers of cells in the lateral and basolateral nuclei. Injections into AIp, PR, and, to a lesser extent, dorsally adjacent neocortical areas produced both retrograde and anterograde labeling in the contralateral amygdala. The main nuclei with contralateral insular and temporal projections are the basomedial nucleus, ventral endopiriform nucleus, and nucleus of the lateral olfactory tract. The contralateral central nucleus and to a lesser extent the lateral nucleus exhibited anterograde labeling. The pattern of retrograde labeling seen with injections at different rostrocaudal levels of the AIp-PR continuum indicates that amygdalocortical projections to these areas exhibit an overlapping topographical organization. Comparison of the results of this study with findings on amygdaloprefrontal cortical efferents suggests that amygdaloid projections to the entire fronto-insulo-temporal mesocortical field are topographically organized.

Autoradiographic localization of mas proto-oncogene mRNA in adult rat brain using in situ hybridization.

The cellular localization and the distribution of the mas proto-oncogene/angiotensin receptor mRNA have been studied in the male rat brain using in situ hybridization with radiolabelled mas cRNA probes. Neuronal cell populations in the forebrain were selectively labelled. A strong specific labelling was demonstrated in the dentate gyrus, the CA3 and CA4 areas of the hippocampus, the olfactory tubercle (medical part), the piriform cortex and the olfactory bulb, while a weak to moderate labelling was present all over the neocortex and especially in the frontal lobe.

Longitudinal changes in injection behaviors in a cohort of injection drug users.

To determine how the injecting behaviors of injection drug users (IDUs) change over time in the context of the epidemic of acquired immunodeficiency syndrome (AIDS) and what factors may be associated with such changes, a cohort of IDUs (n = 313) initially in treatment provided structured interviews regarding drug injecting behaviors. Repeat interviews in 18 months assessed behavior change in subjects who could be contacted for follow-up (n = 220, 70.3%). The study occurred in a state where sterile syringes can be purchased without prescription in drug stores. Injection drug use occurred for 180 (81.8%) of the 220 subjects in the 12 months prior to the initial interview but in only 121 (55.0%) in the 10 months prior to the follow-up interview (p < 0.0001). Similarly, sharing of equipment decreased from 63.1% to 31.8% (p < 0.0001). Sharing with multiple partners declined from 41.9% to 10.6% (p < 0.0001). Factors associated with ongoing risk included use of injected and non-injected psychoactive substances, less time in drug dependence treatment during follow-up interval, having a sexual partner who was an IDU and not using a drug store as the primary source of syringes. Factors associated with multiple-partner sharing included use of psychoactive substances, younger age and nonwhite race.

Pre-treatment characteristics, program philosophy and level of ancillary services as predictors of methadone maintenance treatment outcome.

Predictors of methadone maintenance treatment outcome have not been extensively studied as they relate to variations in program philosophy, nor have such predictors received much examination among recently treated, older cohorts of opioid addicts for whom drug use patterns have changed. Predictors of outcome were examined at 18 months post-treatment entry for 353 admissions to methadone maintenance who received random assignment to one of three counseling conditions: (1) medication only, (2) standard counseling and (3) enhanced services; and one of two contingency conditions: (1) no contingencies, and (2) contingency contracting in a six-cell 3 x 2 design. Subjects in contingency contracting conditions were placed on contingency contracts for positive urine toxicology results and ultimately discharged for unremitting drug use. All subjects completed the Addiction Severity Index (ASI) and provided weekly urine specimens. Predictors of urinalysis results and treatment retention were determined using bivariate and multivariate techniques. Interactions between subject characteristics by experimental condition assignment were also examined as predictors. Higher rates of total positive urine specimens were predicted by younger age, greater pre-treatment frequency of smoking cocaine, lower ASI psychiatric composite scores, and higher ASI legal composite scores. Higher rates of opiate positive specimens were predicted by younger age, lower pre-treatment frequency of alcohol intoxication, higher ASI legal and lower ASI employment and psychiatric composite scores, and assignment to medication only/no contingencies condition. Higher rates of cocaine positives were predicted by younger age, black race, lower ASI psychiatric composite score, greater pre-treatment frequency of intravenous and smoked cocaine use, less pre-treatment frequency of marijuana use, and lower methadone dose level. Assignment to enhanced/contingency contracting predicted lower rates of cocaine positives. Treatment retention was predicted by older age, non-black race, lower ASI legal composite score, higher methadone dose level and assignment to non-contingent conditions. While subject variables over which treatment providers have little control were, thus, related to outcome, type of treatment provided and methadone dose also influenced outcome.

Outcome of a second episode of methadone maintenance.

This paper reports the treatment progress of methadone maintenance clients who were discharged or withdrew from treatment and then were readmitted for a second episode of treatment. Thirty-nine clients in a contingency contract condition remained in treatment long enough (6 months) during both the initial and a second treatment episode, to be exposed to discharge sanctions that were part of the contingency contract. Of these clients 34 failed treatment during the initial treatment episode. Nine (26%) of these initial treatment failures improved their performance in the second episode compared to the first, and only one (20%) of five initial treatment successes who left treatment during their first treatment episode for non-contract reasons showed a poorer performance (failing the second after succeeding in the first episode). Of 17 clients in a condition that applied no contingencies for positive urines, three of 14 (21%) who failed during the initial treatment episode improved their performance, and two of three (67%) who succeeded during the initial treatment episode failed in the second episode. For a subset of clients the efficacy of contingency contracting may not be realized until it is reapplied during a subsequent admission.

Reporting of HIV risk behaviors by injection drug using heterosexual couples in methadone maintenance.

This study sought to identify differences within injection drug using (IDU) couples in reporting of sexual and needle risk behavior. Subjects were thirty-nine heterosexual couples entering methadone maintenance. In 33.3% of couples, one member reported sharing needles while the other member reported no sharing. In 12.9% of couples, one member reported sharing injection equipment, while the other member reported no sharing. Agreement was 77.4% between members of monogamous couples regarding frequency of condom use, 80.7% regarding vaginal intercourse with condoms, and 25.8% regarding vaginal intercourse without condoms. Within couples, a number of differences between members of the couple in injection equipment sharing were noted, suggesting that individuals who attempt to protect themselves by not sharing injection equipment may be placed at risk by their sexual partners. Further clinical and research efforts should be directed toward reducing barriers to behavior that would protect both partners. Implications for self-report measurement of HIV risk behavior and for preventive interventions are discussed.

Cerebellar liponeurocytoma. Case report and review of the literature.

Cerebellar liponeurocytoma is a rare tumor of the posterior fossa that has many morphological similarities to medulloblastoma and neurocytoma. Recently the World Health Organization working group for classification of central nervous system neoplasms adopted the term "cerebellar liponeurocytoma" to provide a unified nomenclature for a tumor variously labeled in the literature as lipomatous medulloblastoma, lipidized medulloblastoma, medullocytoma. neurolipocytoma, lipomatous glioneurocytoma, and lipidized mature neuroectodermal tumor of the cerebellum. The rarity of this tumor and paucity of pertinent information regarding its biological potential and natural history have resulted in the application of various treatment modalities. It is suggested in the available literature that these lesions have a much more favorable prognosis than typical medulloblastomas, and that adjuvant therapy for liponeurocytoma need not be as extensive as that administered for medulloblastomas.

Optimizing accuracy in magnetic resonance imaging-guided stereotaxis: a technique with validation based on the anterior commissure-posterior commissure line.

OBJECT: Some of the earliest successful frame-based stereotactic interventions directed toward the thalamus and basal ganglia depended on identifying the anterior commissure (AC) and posterior commissure (PC) in a sagittal ventriculogram and defining the intercommissural line that connects them in the midsagittal plane. The AC-PC line became the essential landmark for the localization of neuroanatomical targets in the basal ganglia and diencephalon and for relating them to stereotactic atlases. Stereotactic/functional neurosurgery has come to rely increasingly on magnetic resonance (MR) imaging guidance, and methods for accurately determining the AC-PC line on MR imaging are being developed. The goal of the present article is to present the authors' technique. METHODS: The technique described uses MR sequences that minimize geometric distortion and registration error, thereby maximizing accuracy in AC-PC line determinations from axially displayed MR data. The technique is based on the authors' experience with the Leksell G-frame but can be generalized to other MR imaging-based stereotactic systems. This methodology has been used in a series of 62 stereotactic procedures in 47 adults (55 pallidotomies and seven thalamotomies) with preliminary results that compare favorably with results reported when using microelectrode recordings. The measurements of the AC-PC line reported here also compare favorably with those based on ventriculography and computerized tomography scanning. CONCLUSIONS: The methodology reported here is critical in maintaining the accuracy and utility of MR imaging as its role in modern stereotaxy expands. Accurate parameters such as these aid in ensuring the safety, efficacy, and reproducibility of MR-guided stereotactic procedures.

Users of free treatment slots at a community-based methadone maintenance clinic.

At a community-based methadone clinic in Seattle, WA, 360 opiate-addicted individuals were enrolled in a treatment demonstration project. The treatment slots were free to clients and, unlike other funded treatment slots, did not require proof of eligibility based on documentation of indigence. The clients were compared with 70 clients enrolled in a research project begun 2 years earlier in which the sample was drawn from a population using normal funding sources at the same program. Subjects in the later demonstration project were older and had fewer years of education. A higher percentage of the demonstration project subjects were African American. These differences indicate that introduction of free treatment opened opportunities to individuals who have difficulty accessing treatment under normal circumstances. Subjects in the treatment demonstration project were more likely to have obtained needles from legal sources and used bleach to clean needles. These findings probably reflect the impact of needle exchange and outreach programs, established in the year prior to the demonstration project.

A comparison of two aversion treatment methods for alcoholism.

In a follow-up study, patients who had received either chemical or electrical aversion treatment reported similar rates of abstinence.

Contingency management of urinalysis results and intensity of counseling services have an interactive impact on methadone maintenance treatment outcome.

In a 3 x 2 factorial design, 360 new admissions to methadone maintenance were randomly assigned to one of three levels of counseling: (1) "medication only," (2) "standard" counseling, and (3) "enhanced" services; and one of two contingency contracting conditions: (1) no contingencies (NC), and (2) contingency contracting (CC). Contingency contracting included discharge for continuous positive urines; subsequently CC subjects were discharged at a greater rate than the NC group. However, CC subjects were more likely to be readmitted. NC subjects provided more urines positive for any illicit drug use than did CC subjects. For opiate positives a significant level of counseling by contingency contracting interaction was found with medication only/CC subjects obtaining fewer opiate positives than medication only/NC subjects. The impact of reduced or enhanced services and of contingency contracting will not be fully understood until longer term follow-up (18 and 24 month) is completed. Results suggest that contingency management procedures could be utilized in settings offering minimum services (e.g., "interim methadone") to achieve treatment outcomes similar to programs offering standard counseling services.