A dataset of global ocean alkaline phosphatase activity.

Utilisation of dissolved organic phosphorus (DOP) by marine microbes as an alternative phosphorus (P) source when phosphate is scarce can help sustain non-Redfieldian carbon:nitrogen:phosphorus ratios and efficient ocean carbon export. However, global spatial patterns and rates of microbial DOP utilisation are poorly investigated. Alkaline phosphatase (AP) is an important enzyme group that facilitates the remineralisation of DOP to phosphate and thus its activity is a good proxy for DOP-utilisation, particularly in P-stressed regions. We present a Global Alkaline Phosphatase Activity Dataset (GAPAD) with 4083 measurements collected from 79 published manuscripts and one database. Measurements are organised into four groups based on substrate and further subdivided into seven size fractions based on filtration pore size. The dataset is globally distributed and covers major oceanic regions, with most measurements collected in the upper 20 m of low-latitude oceanic regions during summer since 1997. This dataset can help support future studies assessing global ocean P supply from DOP utilisation and provide a useful data reference for both field investigations and modelling activities.

Development of the 1st International Reference Panel for HIV-1 RNA genotypes for use in nucleic acid-based techniques.

Twenty-eight laboratories from 16 countries participated in a collaborative study to evaluate an HIV-1 RNA Genotype Reference Panel for use with nucleic acid-based tests (NAT). The Reference Panel consisted of 11 coded samples representing different HIV-1 genotypes (subtypes A-D, AE, F, G, AA-GH, groups N and O) as well as a negative diluent control. Each laboratory assayed the eleven panel members concurrently with the 1st International Standard for HIV-1 RNA (NIBSC Code 97/656) on at least three separate occasions and the data collated and analysed at NIBSC. Twenty-nine sets of data from NAT were received, 19 from quantitative and 10 from qualitative assays, with six different commercial assays and five "in-house" assays represented. The results showed that viruses from subtypes A-D and recombinant virus AE [CRF01_AE] were detected consistently, but that some assays had difficulty with the detection and quantification of viruses from subtypes F and G, a mixed recombinant virus AA-GH and a representative of group N. Furthermore, most assays failed to detect the group O representative. The study illustrated the limitations of some molecular assays particularly in detection of certain non-B genotypes which are important viruses in the global AIDS pandemic and illustrated the value of a well-characterised genotype panel. The panel has been established by the World Health Organisation's Expert Committee on Biological Standardisation as the 1st International Reference Panel HIV-1 RNA Genotypes (code 01/466).

Identification and biological characterization of 6-aryl-7-isopropylquinazolinones as novel TRPV1 antagonists that are effective in models of chronic pain.

Vanilloid receptor 1 (VR1, TRPV1) is a cation-selective ion channel that is expressed on primary afferent neurons and is upregulated following inflammation and nerve damage. Blockers of this channel may have utility in the treatment of chronic nociceptive and neuropathic pain. Here, we describe the optimization from a high throughput screening hit, of a series of 6-aryl-7-isopropylquinazolinones that are TRPV1 antagonists in vitro. We also demonstrate that one compound is active in vivo against capsaicin-induced hyperalgesia and in models of neuropathic and nociceptive pain in the rat.

Vanilloid receptor TRPV1, sensory C-fibers, and vascular autoregulation: a novel mechanism involved in myogenic constriction.

Myogenic constriction describes the innate ability of resistance arteries to constrict in response to elevations in intraluminal pressure and is a fundamental determinant of peripheral resistance and, hence, organ perfusion and systemic blood pressure. However, the receptor/cell-type that senses changes in pressure on the blood vessel wall and the pathway that couples this to constriction of vascular smooth muscle remain unclear. In this study, we show that elevation of intraluminal transmural pressure of mesenteric small arteries in vitro results in a myogenic response that is profoundly suppressed following ablation of sensory C-fiber activity (using in vitro capsaicin desensitization resulted in 72.8+/-10.3% inhibition, n=8; P<0.05). Activation of C-fiber nerve endings by pressure was attributable to stimulation of neuronal vanilloid receptor, TRPV1, because blockers of this channel, capsazepine (71.9+/-11.1% inhibition, n=9; P<0.001) and ruthenium red (46.1+/-11.7% inhibition, n=4; P<0.05), suppressed the myogenic constriction. In addition, this C-fiber dependency is likely related to neuropeptide substance P release and activity because blockade of tachykinin NK1 receptors (66.3+/-13.7% inhibition, n=6; P<0.001), and not NK2 receptors (n=4, NS), almost abolished the myogenic response. Previous studies support a role for 20-hydroxyeicosatetraenoic acid (20-HETE) in myogenic constriction responses; herein, we show that 20-HETE-induced constriction of mesenteric resistance arteries is blocked by capsazepine. Together, these results suggest that elevation of intraluminal pressure is associated with generation of 20-HETE that, in turn, activates TRPV1 on C-fiber nerve endings resulting in depolarization of nerves and consequent vasoactive neuropeptide release. These findings identify a novel mechanism contributing to Bayliss' myogenic constriction and highlights an alternative pathway that may be targeted in the therapeutics of vascular disease, such as hypertension, where enhanced myogenic constriction plays a role in the pathogenesis.

Antihyperalgesic activity of a novel nonpeptide bradykinin B1 receptor antagonist in transgenic mice expressing the human B1 receptor.

We describe the properties of a novel nonpeptide kinin B1 receptor antagonist, NVP-SAA164, and demonstrate its in vivo activity in models of inflammatory pain in transgenic mice expressing the human B1 receptor. NVP-SAA164 showed high affinity for the human B1 receptor expressed in HEK293 cells (K(i) 8 nM), and inhibited increases in intracellular calcium induced by desArg10kallidin (desArg10KD) (IC50 33 nM). While a similar high affinity was observed in monkey fibroblasts (K(i) 7.7 nM), NVP-SAA164 showed no affinity for the rat B1 receptor expressed in Cos-7 cells. In transgenic mice in which the native B1 receptor was deleted and the gene encoding the human B1 receptor was inserted (hB1 knockin, hB1-KI), hB1 receptor mRNA was induced in tissues following LPS treatment. No mRNA encoding the mouse or human B1 receptor was detected in mouse B1 receptor knockout (mB1-KO) mice following LPS treatment. Freund's complete adjuvant-induced mechanical hyperalgesia was similar in wild-type and hB(1)-KI mice, but was significantly reduced in mB1-KO animals. Mechanical hyperalgesia induced by injection of the B1 agonist desArg10KD into the contralateral paw 24 h following FCA injection was similar in wild-type and hB1-KI mice, but was absent in mB1-KO animals. Oral administration of NVP-SAA164 produced a dose-related reversal of FCA-induced mechanical hyperalgesia and desArg10KD-induced hyperalgesia in hB1-KI mice, but was inactive against inflammatory pain in wild-type mice. These data demonstrate the use of transgenic technology to investigate the in vivo efficacy of species selective agents and show that NVP-SAA164 is a novel orally active B1 receptor antagonist, providing further support for the utility of B1 receptor antagonists in inflammatory pain conditions in man.

N-Formyl peptide receptor subtypes in human neutrophils activate L-plastin phosphorylation through different signal transduction intermediates.

We investigated the coupling of the fMLP (N -formyl-L-methionyl-L-leucyl-L-phenylalanine; 'chemotactic peptide') receptor with phosphorylation of the actin-binding protein L-plastin in neutrophils. Using two-dimensional IEF (isoelectric focusing)/PAGE and MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight)-MS, L-plastin was identified as a major phosphoprotein in fMLP-stimulated neutrophils whose phosphorylation was dependent on phosphoinositide 3-kinase, PLD (phospholipase D) and PKC (protein kinase C) activity. Two fMLP receptor subtypes were identified in neutrophils, characterized by a distinct sensitivity to fMLP and antagonistic peptides. Both receptor subtypes induced the phosphorylation of L-plastin. L-plastin phosphorylation induced by low-affinity fMLP receptors involves an action of phosphoinositide 3-kinase, PLD and PKC isotypes. In contrast, none of these intermediates are utilized by high-affinity fMLP receptors in the phosphorylation of L-plastin. However, the PKC inhibitor Ro-31-8220 inhibits L-plastin phosphorylation induced by the high-affinity fMLP receptor. Thus, an as yet unknown Ro-31-8220-sensitive kinase regulates L-plastin phosphorylation in response to the high-affinity fMLP receptor. The results suggest a model in which receptor subtypes induce a similar endpoint event through different signal-transduction intermediates. This may be relevant in the context of cell migration in which one receptor subpopulation may become desensitized in a concentration gradient of chemoattractant.

Cloning and functional characterization of the guinea pig vanilloid receptor 1.

We have cloned a guinea pig Vanilloid receptor 1 (VR1) from a dorsal root ganglion cDNA library and expressed it in CHO cells. The receptor has been functionally characterized by measuring changes in intracellular calcium produced by capsaicin, low pH and noxious heat. Capsaicin produced a concentration-dependent increase in intracellular calcium in guinea pig VR1-CHO cells with an estimated EC(50) of 0.17 +/- 0.0065 micro M, similar to that previously reported for rat and human VR1. Olvanil and resiniferatoxin were also effective agonists (EC(50) values of 0.0087 +/- 0.0035 micro M and 0.067 +/- 0.014 micro M, respectively), but 12-phenylacetate 13-acetate 20-homovanillate (PPAHV) and anandamide showed little agonist activity up to 10 micro M. As with human and rat VR1, guinea pig VR1 was also activated by pH below 6.0 and by noxious heat (>42 degrees C). Capsazepine acted as an antagonist of capsaicin responses in guinea pig VR1-CHO cells (IC(50) of 0.324 +/- 0.041 micro M ), as seen at rat VR1. However, in contrast to its lack of activity against pH and heat responses at rat VR1, capsazepine was an effective antagonist of these responses at guinea pig VR1. Capsazepine displayed an IC(50) of 0.355 +/- 25 micro M against pH 5.5, and provided complete blockade of heat responses at 1 micro M. Thus, capsazepine can significantly inhibit calcium influx due to heat and pH 5.5 at guinea pig VR1 and human VR1 but is inactive against these activators at rat VR1.

Potent and orally bioavailable non-peptide antagonists at the human bradykinin B(1) receptor based on a 2-alkylamino-5-sulfamoylbenzamide core.

The bradykinin B(1) receptor is rapidly induced after inflammation or tissue trauma and appears to play an important role in the maintenance of hyperalgesia in inflammatory conditions. Here, we describe the optimization process to identify novel, potent non-peptide human B(1) receptor antagonists based on a 2-alkylamino-5-sulfamoylbenzamide core. Optimized derivatives are selective, functional B(1) antagonists with low nanomolar affinity and exhibit oral bioavailability in animals.

Nonpeptide bradykinin B2 receptor antagonists: conversion of rodent-selective bradyzide analogues into potent, orally-active human bradykinin B2 receptor antagonists.

The 1-(2-nitrophenyl)thiosemicarbazide (TSC) derivative, (S)-1-[4-(4-benzhydrylthiosemicarbazido)-3-nitrobenzenesulfonyl]pyrrolidine-2-carboxylic acid [2-[(2-dimethylaminoethyl)methylamino]ethyl]amide (bradyzide; (S)-4), was recently disclosed as a novel, potent, orally active nonpeptide bradykinin (BK) B2 receptor antagonist. The compound inhibited the specific binding of [3H]BK to NG108-15 cell membrane preparations (rodent neuroblastoma-glioma) expressing B2 receptors with a K(i) of 0.5 +/- 0.2 nM. Compound (S)-4 also demonstrated oral efficacy against Freund's complete adjuvant (FCA)-induced mechanical hyperalgesia in rats with an ED50 value of 0.84 micromol/kg. After we optimized the terminal binding determinants projecting from the TSC framework, we found that it was possible to replace the potentially toxicophoric nitro and divalent sulfur moieties with only a 15-fold loss in binding affinity ((S)-14a). However, bradyzide and its congeners were found to have much lower affinities for cloned human B2 receptors, expressed in Cos-7 cells. The hitherto synthesized TSC series was screened against the human B2 receptor, and the dibenzosuberane (DBS) pharmacophore emerged as the key structural requirement for potency. Incorporation of this group resulted in a series of derivatives ((S)-14d,e and 19b-d) with K(i) ranges of 10.7-176 nM in NG108-15 cells (expressing the rodent B2 receptor) and 0.79-253 nM in Cos-7 cells (expressing the human B2 receptor). There was no evidence of agonist activity with any of the nonpeptides in any of the cell lines tested. In vivo, oral administration of compound 19c reversed FCA-induced and turpentine-induced mechanical hyperalgesia in rodents with ED50 values of 0.027 and 0.32 micromol/kg, respectively. The selectivity profiles of compounds (S)-14f and (S)-14g were also assessed to determine the conformational and/or steric preferences of the double-ring arrangement. The affinity of (S)-14 g for the human B2 receptor suggested that it may be a hydrophobic interaction with the ethane bridge of the DBS moiety that accounts for the increased potency of compounds (S)-14d,e and 19b,c at this receptor, by favoring a binding mode inaccessible to the unsubstituted diphenylmethyl derivative, (S)-4.

Calibration of HIV-1 working reagents for nucleic acid amplification techniques against the 1st international standard for HIV-1 RNA.

Many laboratories use working reagents/run controls to monitor the performance of their nucleic acid amplification techniques (NAT) for the measurement of HIV-1 RNA. A collaborative study was carried out in order to calibrate seven internationally available working reagents, QC105 (National Serology Reference Laboratory [NRL], Australia), B5 and B10 (Center for Biological Evaluation and Research [CBER], USA), Pelispy (Central Laboratory of the Netherlands Blood Transfusion Service [CLB], The Netherlands), PWS-1 and PWS-3 (National Institute for Biological Standards and Control [NIBSC], UK) and IRC (Virology Networks [VN], The Netherlands) against the 1st International Standard for HIV-1 RNA (code 97/656). Twenty-one laboratories from 12 different countries participated in the collaborative study and from the results it was determined that QC105 contained 4.0 log(10) International Units (IU)/ml, B5 2.2 log(10) IU/ml, B10 3.8 log(10) IU/ml, Pelispy 4.4 log(10) IU/ml, PWS-1 3.6 log(10) IU/ml, PWS-3 2.7 log(10) IU/ml and IRC 4.3 log(10) IU/ml. The seven working reagents calibrated in this international study may be used to validate and standardise the large number of qualitative and quantitative, commercial and in-house NAT assays that are currently being applied in the fields of blood safety and patient management. They will also help laboratories to comply with the sensitivity requirements that may be brought in by the regulatory authorities and may contribute to further harmonisation of guidelines on NAT published by organisations such as the European Medicines Evaluation Agency (EMEA), Paul-Ehrlich Institute and CBER, FDA.

Detection and quantitation of human immunodeficiency virus type-1 particles by confocal microscopy.

A method is described to visualise directly human immunodeficiency virus type-1 (HIV-1) particles. HIV-1 containing samples were adsorbed onto a plastic surface and doubly labeled with antibodies specific for viral proteins and sensitive nucleic acids dyes. Laser scanning confocal microscopy detected co-localization of viral proteins and nucleic acids, thus allowing specific identification of HIV. Using this technique, we have quantified eight different HIV-1 sub-types and three HIV-1 groups in tissue culture supernatants from infected peripheral blood mononuclear cells (PBMCs). Confocal counts correlated well with electron microscopy (EM) counts and HIV-1 RNA loads as determined by quantitative PCR. Confocal microscopy may prove to be a simple alternative to electron microscopy for virus identification and quantitation.

The pharmacology of T-kinin and des-Arg(11)-T-kinin in primary cultures of rat bladder smooth muscle cells.

T-kinin and its putative carboxypeptidase product des-Arg(11)-T-kinin are members of the kinin family that are unique to the rat. Primary cultures of rat bladder smooth muscle cells were used to investigate the pharmacology of these peptides. Calcium imaging experiments showed that rat bladder smooth muscle cells responded to both bradykinin and des-Arg(9)-bradykinin with an increase in [Ca(2+)](i) and responses to both agonists could be observed in the same cell. A more detailed pharmacological characterisation with a range of bradykinin receptor agonists and antagonists using 45Ca(2+) efflux confirmed the presence of both B(1) and B(2) bradykinin receptors. Using this cellular model, we confirm that T-kinin is a bradykinin B(2) receptor agonist and show for the first time that des-Arg(11)-T-kinin is a potent and selective bradykinin B(1) receptor agonist. In addition, using cells expressing the cloned rat and human bradykinin B(2) receptors plus the Ca(2+)-sensitive protein aequorin, T-kinin was shown to be selective for the rat over the human bradykinin B(2) receptor.

Journal club: Integrating research awareness into postgraduate nurse training.

BACKGROUND: Evidence-based nursing requires nurses to maintain an awareness of recently published research findings to integrate into their clinical practice. In the South African setting keeping up with recent literature has additional challenges, including the diversity of nurses' home language, geographically foreign origins of published work, and limited economic resources. Students enrolled in a postgraduate programme came from various paediatric settings and displayed limited awareness of nursing literature as an evidence base for practice. OBJECTIVES: The study aimed to design and introduce a journal club as an educational strategy into the postgraduate programmes in children's nursing at the University of Cape Town (UCT), and then to refine the way it is used to best serve programme outcomes and facilitate student learning whilst still being an enjoyable activity. METHOD: An action research methodology using successive cycles of 'assess-plan-act-observe' was used to design, implement and refine the structure of a journal club within the postgraduate diploma programme over four academic years. Six educators actively tracked and reflected on journal club sessions, and then analysed findings during and after each annual cycle to plan improvement and increasing programme alignment. RESULTS: Considerable refinement of the intervention included changing how it was structured, the preparation required by both students and educators, the article selection process and the intervention's alignment with other learning activities in the programme. CONCLUSION: Journal club facilitated an increase in student awareness and reading of nursing literature, offering the opportunity to consider application of published research to current nursing practice. Another benefit was enabling students to become familiar with the specialised and technical language of research, children's nursing and the critical care of children and neonates, by speaking about these in peer settings.

Utility of the Rosenberg self-esteem scale.

The Rosenberg Self-Esteem Scale (RSES) continues to be used to purportedly measure self-esteem of people with intellectual disabilities, despite the lack of sound evidence concerning its validity and reliability when employed with this population. The psychometric foundations of the RSES were analyzed here with a sample of 219 participants with intellectual disabilities. The factor analytic methods employed revealed two factors (Self-Worth and Self-Criticism) and more specific problems with RSES Items 5 and 8. Overall, this scale showed only moderate temporal and moderate internal reliability and poor aspects of criterion validity. Results are discussed with reference to either developing a new measure of self-esteem or redesigning and simplifying the RSES in order to increase its initial face validity in intellectual disability samples.

Mitochondrial DNA and retroviral RNA analyses of archival oral polio vaccine (OPV CHAT) materials: evidence of macaque nuclear sequences confirms substrate identity.

Inoculation of live experimental oral poliovirus vaccines (OPV CHAT) during the 1950s in central Africa has been proposed to account for the introduction of HIV into human populations. For this to have occurred, it would have been necessary for chimpanzee rather than macaque kidney epithelial cells to have been included in the preparation of early OPV materials. Theoretically, this could have led to contamination with a progenitor of HIV-1 derived from a related simian immunodeficiency virus of chimpanzees (SIVCPZ). In this article we present further detailed analyses of two samples of OPV, CHAT 10A-11 and CHAT 6039/Yugo, which were used in early human trials of poliovirus vaccination. Recovery of poliovirus by culture techniques confirmed the biological viability of the vaccines and sequence analysis of poliovirus RNA specifically identified the presence of the CHAT strain. Independent nested sets of oligonucleotide primers specific for HIV-1/SIVCPZ and HIV-2/SIVMAC/SIVSM phylogenetic lineages, respectively, indicated no evidence of HIV/SIV RNA in either vaccine preparation, at a sensitivity of 100 RNA equivalents/ml. Analysis of cellular substrate by the amplification of two distinct regions of mitochondrial DNA (D-loop control region and 12S ribosomal sequences) revealed no evidence of chimpanzee cellular sequences. However, this approach positively identified rhesus and cynomolgus macaque DNA for the CHAT 10A-11 and CHAT 6039/Yugo vaccine preparations, respectively. Analysis of multiple clones of mtDNA 12S rDNA indicated a relatively high number of nuclear mitochondrial DNA sequences (numts) in the CHAT 10A-11 material, but confirmed the macaque origin of cellular substrate used in vaccine preparation. These data reinforce earlier findings on this topic providing no evidence to support the contention that poliovirus vaccination was responsible for the introduction of HIV into humans and sparking the AIDS pandemic.