Use of F-Specific RNA Bacteriophage to Estimate Infectious Norovirus Levels in Oysters.

Contamination of bivalve shellfish, particularly oysters, with norovirus is recognised as a significant food safety risk. Methods for quantification of norovirus in oysters using the quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) are well established, and various studies using RT-qPCR have detected norovirus in a considerable proportion of oyster samples, both in the UK and elsewhere. However, RT-qPCR detects viral genome, and by its nature is unable to discriminate between positive results caused by infectious viruses and those caused by non-infectious remnants including damaged virus particles and naked RNA. As a result, a number of alternative or complementary approaches to RT-qPCR testing have been proposed, including the use of infectious viral indicator organisms, most frequently F-specific RNA bacteriophage (F-RNA phage). In this study, we investigated the relationships between F-RNA phage and norovirus in digestive tissues from two sets of oyster samples, one randomly collected at retail (630 samples), and one linked to suspected norovirus illness outbreaks (nine samples). A positive association and correlation between PCR-detectable levels of genogroup II F-RNA bacteriophage (associated with human faecal contamination) and norovirus was found in both sets of samples, with more samples positive for genogroup II phage, at generally higher levels than norovirus. Levels of both viruses were higher in outbreak-related than retail samples. Infectious F-RNA phage was detected in 47.8% of all retail samples, and for a subset of 224 samples where characterisation of phage was carried out, infectious GII phage was detected in 30.4%. Infectious GII phage was detected in all outbreak-related samples. Determination of infectivity ratios by comparing levels of PCR-detectable (copies/g) and infectious GII phage (pfu/g) revealed that in the majority of cases less than 10% of virus detected by RT-qPCR was infectious. Application of these ratios to estimate infectious norovirus levels indicated that while 77.8% of outbreak-related samples contained > 5 estimated infectious norovirus/g, only 13.7% of retail samples did. Use of a combination of levels of PCR-detectable norovirus and infectious F-RNA phage showed that while only 7.0% of retail samples contained both > 100 copies/g norovirus and > 10 pfu/g F-RNA phage, these combined levels were present in 77.8% of outbreak-related samples, and 75.9% of retail samples with > 5 estimated infectious norovirus/g. We therefore suggest that combining RT-qPCR testing with a test for infectious F-RNA phage has the potential to better estimate health risks, and to better predict the presence of infectious norovirus than RT-qPCR testing alone.

Microxine, a new cdc2 kinase inhibitor from the Australian marine sponge Microxina species.

A new purine derivative microxine (1) was isolated from the Australian marine sponge Microxina sp. The compound was isolated via reversed-phase chromatography and its structure determined spectroscopically. Microxine was found to weakly inhibit cdc2 kinase activity with an IC(50) of 13 microM.

New anilinophthalazines as potent and orally well absorbed inhibitors of the VEGF receptor tyrosine kinases useful as antagonists of tumor-driven angiogenesis.

The sprouting of new blood vessels, or angiogenesis, is necessary for any solid tumor to grow large enough to cause life-threatening disease. Vascular endothelial growth factor (VEGF) is one of the key promoters of tumor induced angiogenesis. VEGF receptors, the tyrosine kinases Flt-1 and KDR, are expressed on vascular endothelial cells and initiate angiogenesis upon activation by VEGF. 1-Anilino-(4-pyridylmethyl)-phthalazines, such as CGP 79787D (or PTK787 / ZK222584), reversibly inhibit Flt-1 and KDR with IC(50) values < 0.1 microM. CGP 79787D also blocks the VEGF-induced receptor autophosphorylation in CHO cells ectopically expressing the KDR receptor (ED(50) = 34 nM). Modification of the 1-anilino moiety afforded derivatives with higher selectivity for the VEGF receptor tyrosine kinases Flt-1 and KDR compared to the related receptor tyrosine kinases PDGF-R and c-Kit. Since these 1-anilino-(4-pyridylmethyl)phthalazines are orally well absorbed, these compounds qualify for further profiling and as candidates for clinical evaluation.

Identification of a high-affinity anti-phosphoserine antibody for the development of a homogeneous fluorescence polarization assay of protein kinase C.

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 +/- 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO(4))LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO(4))LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca(2+)-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.

Pediatric Milliman and Robertson length-of-stay criteria: are they realistic?

OBJECTIVE: Guidelines for inpatient length of stay (LOS) have been developed by several groups; among the most widely applied are those published by Milliman and Robertson (M&R). Few published reports have examined the relationship of actual practice to such guidelines, none in pediatric populations. This study was designed to compare pediatric practice in a large and defined population to M&R LOS criteria. METHODS: Administrative data from New York State in 1995 were used to examine LOS for discharges corresponding to 16 selected pediatric diagnoses for which M&R publishes guidelines. Outliers, defined as the 2% of discharges with the longest LOS, were eliminated. The distribution of LOS for each diagnosis was compared with M&R LOS guidelines. RESULTS: In New York State during 1995, pediatric LOS was markedly divergent from M&R guidelines. In general, the percentage of discharges in excess of the criterion LOS was less for nonmandatory admissions (croup: 23%, gastroenteritis: 44%, and pneumonia: 48%) than for those requiring surgery (uncomplicated appendectomy: 67%, pyloromyotomy: 62%, and major but noncritical burns: 64%) or prolonged treatment with antibiotics (bacterial meningitis: 91% and osteomyelitis: 86%). CONCLUSIONS: In New York State during 1995, LOS for selected pediatric conditions was generally in excess of published M&R guidelines. This raises concern about the potential effects of such guidelines on both patients and the hospitals caring for them. While endorsing the need for cost-effective practice, we call attention to the methods used to develop and validate guidelines.length of stay, pediatrics, managed health care, administrative data, practice guidelines.

The associations among pediatricians' knowledge, attitudes, and practices regarding emergency contraception.

OBJECTIVES: To quantify practitioner administration of the emergency contraceptive pill (ECP) among adolescent patients, and to determine if such administration is associated with physician knowledge and attitudes regarding efficacy, side effects, and appropriate use. DESIGN: Survey of pediatricians. SETTING: The survey address list was generated from a database of active Fellows of the American Academy of Pediatrics in the District of Columbia metropolitan area. MAIN OUTCOMES MEASURES: Prescription of the ECP in the previous 12 months, or counseling of an adolescent patient about the ECP. RESULTS: Of the 236 questionnaires distributed, 143 (61%) were returned and 121 (51%) were usable. Twenty-four pediatricians (20%) reported prescribing the ECP, and 29 (24%) had counseled adolescent patients about the ECP. Of the practice-related variables surveyed, both the number of adolescents seen per week and the practice setting were significantly associated with these outcomes. Of the knowledge-related variables surveyed, knowledge of the timing and the Food and Drug Administration-labeled status of the ECP were significantly associated with outcomes. None of the attitude-related variables surveyed were associated with outcomes. CONCLUSIONS: This study demonstrates that knowledge deficits, not attitude-related variables, are significantly associated with the low level of ECP administration and counseling among District of Columbia pediatricians. Because knowledge deficits are amenable to educational interventions, our data suggest that informing pediatricians about the ECP may increase its administration among their adolescent patients.emergency contraceptive pill, pediatricians, adolescents.

Measurement of cdk4 kinase activity using an affinity peptide-tagging technology.

Cyclin-dependent kinases such as Cdk4 are involved in the control of cell cycle progression, and misregulation of Cdk4 has been implicated in many types of cancers. In the present study, we report the development of a novel homogeneous assay using an affinity peptide-tagging technology for rapidly discovering Cdk4 inhibitors. The DNA sequence encoding a streptavidin recognition motif, or StrepTag (AWRHPQFGG), was cloned and expressed at the C-terminus of a fusion protein of a 152-amino acid hyperphosphorylation domain (Rb152) of the retinoblastoma protein (Rb) linked to GST at the N-terminus. This affinity peptide-tagged protein (GST-Rb152-StrepTag), which contains the two known phosphorylation sites of Rb, specifically phosphorylated by Cdk4 in vivo, was used as a substrate in the current in vitro kinase assay. After phosphorylation, scintillation proximity assay (SPA) scintillant beads coated with streptavidin were added. Radiolabeled GST-Rb152-StrepTag was brought in close proximity to the SPA scintillant beads through the interaction between StrepTag and streptavidin, resulting in the emission of light from beads. By applying the affinity peptide-tagging technology, we have eliminated the separation and wash steps which are normally required in a radioactive filtration assay. Therefore, this homogeneous method is simple, robust, and highly amenable to high-throughput screening of Cdk4-specific inhibitors. Furthermore, the affinity peptide tagging technique reported here is a simple, generic method that can be applied to many recombinant proteins for the development of kinase and protein-protein interaction assays.

Partnerships in healthcare: a Commonwealth perspective.

This article briefly outlines the nature and characteristics of partnership, and points to the need to build partnerships through deliberate efforts, since they do not occur by simply bringing people and/or resources together. It identifies some of the reasons why partnerships in health are invaluable in meeting the priority health needs of populations, and the importance which the Commonwealth places on partnerships given the unique characteristics which are common to its member countries. The article then uses practical examples to illustrate the value of partnerships in health at the national, regional and international level. For the benefit of front-line healthcare providers, some suggestions are given of how partnerships could be used to enhance the services which they give to the communities they serve.

Novel C5a receptor antagonists regulate neutrophil functions in vitro and in vivo.

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1-71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1-71 derivatives with replacements of Q71, C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various lengths (71-74) with Q71C replacements. The majority of C5a1-71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1-71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1-71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, CSaRAM and CSaRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 microM), and chemotaxis (11 and 7 microM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.

Prolonged hypoglycemic crisis associated with glyburide.

An elderly patient taking glyburide 5 mg/day for noninsulin-dependent diabetes mellitus was hospitalized because of severe hypoglycemia. Laboratory results indicated both renal and hepatic abnormalities were present at the time of admission. Despite infusion of 10% dextrose and supplemental boluses of 50% dextrose, the hypoglycemic crisis persisted for 3 days. After it resolved, the patient's diabetes was controlled by diet alone. The patient's age and the presence of hepatic and/or renal impairment must be taken in account in prescribing glyburide. Recognizing patients who may require dosage changes, and educating them on the signs and symptoms of hypoglycemia, may help prevent hospitalizations resulting from this complication associated with glyburide.

In vitro and in vivo evaluation of an endothelin inhibitor reveals novel K+ channel opening activity.

A low molecular weight endothelin (ET-1) inhibitor (Ex. 127, European Patent Application 404 525 A2, Takeda Chemical Ind., 1991), CGS 26061, was synthesized and evaluated to determine its mechanism of action. CGS 26061 (10 microM) failed to inhibit binding of [125I]ET-1 in porcine thoracic aorta and was without effect on ET-1-induced [3H]inositol phosphate accumulation in A7r5 cells. However, CGS 26061 relaxed porcine coronary arterial rings precontracted with ET-1. In addition, contractions to PGF2 alpha and low K+ (20 mM) but not high K+ were attenuated, suggesting that CGS 26061 (1, 10 microM) is a potassium channel opener. Patch-clamp experiments confirmed the K+ channel activity (0.1-10 microM). The originally re ported inhibition of ET-1-induced pressor responses by Ex. 127 (CGS 26061) was not replicated in the anesthetized dog or conscious rat nor was it shown to be antihypertensive in SHR. These data have identified CGS 26061 as a novel K+ channel opener with a unique cardiovascular profile.

Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates.

A nonradioactive, sensitive assay method to evaluate the activity of protein tyrosine kinases is described. This method utilizes europium chelate-labeled anti-phosphotyrosine antibodies to detect phosphate transfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissociation-enhanced fluorescence. Recombinant c-src was used to demonstrate that substrate phosphorylation was dependent on incubation time, enzyme concentration, and the amount of substrate used to coat the microtiter plate wells. A series of proprietary c-src inhibitors was evaluated in competition assays, and demonstrated a rank order of potency which was identical to that determined by other assay methods. Substrate phosphorylation was also demonstrated to be dependent on the concentration of ATP present during the kinase reaction. Because the kinase assay can be performed with different ATP concentrations (unlike with assays utilizing radioactive ATP analogs), the assay described can be used to distinguish compounds that compete for the ATP or substrate binding sites of the kinase.

Characterization of an interleukin 6 cytokine family antagonist protein from a marine sponge, Callyspongia sp.

An inhibitor of IL-6 binding to the human hepatoma line HepG2 and myeloma cell line U266 was identified in a saline extract of the marine sponge, Callyspongia sp. Functional activity, measured through the increase in haptoglobin production by HepG2 cells stimulated with IL-6, could be strongly inhibited by the extract. Similarly, IL-6-induced production of IgM by the B cell line SKW6.4 was substantially reduced. In neither cell line was there evidence of toxicity produced by the extract. Other sponges of the Callyspongia species were found to contain analogous activity. The activity was destroyed by trypsin treatment or boiling of the extract, suggesting that the inhibition is due to a protein. When the binding of IL-6 to its receptor complex was dissected in vitro, inhibition of binding of IL-6 to soluble receptor by the extract was not detected, but binding of the IL-6-sIL-6R complex to soluble gp130 was inhibited in a dose-dependent fashion. This was borne out in cellular assays since the extract inhibited activation of HepG2 cells stimulated with oncostatin M or leukemia inhibitory factor, cytokines which also use gp130 for signal transduction. These results suggest that the Callyspongia extract contains a protein which blocks the interaction of the IL-6 family of cytokines with their signal transduction moiety, gp130. Elucidation of the structure and mode of action of such a protein would be helpful in designing gp130 antagonists to inhibit the functions of this cytokine family, overproduction of which has been associated with cancer and pathologies of autoimmune disease and AIDS.

A solid-phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates.

A solid-phase assay for the determination of protein tyrosine kinase (PTK) activity has been developed. The transfer of 33PO4 from ATP to the synthetic substrate poly(Glu, Tyr) 4:1 attached to the bioactive surface of scintillating microtiter plates served as the basis to evaluate enzyme activity. The procedure eliminates detection with phosphotyrosine antibodies, tedious separation of phosphorylated peptides with phosphocellulose membranes, and extensive washing steps. For these reasons, the traditionally time-consuming procedure can be performed with a simple three-step protocol. The method is highly accurate, rapid, and robotics friendly. The advantages over existing assays make this procedure especially suited for high throughput applications.