Mapping the CGRP receptor ligand binding domain: Tryptophan-84 of RAMP1 is critical for agonist and antagonist binding

The calcitonin receptor-like receptor (CLR) associates with the accessory protein RAMP1 to form a receptor for the neuropeptide calcitonin gene-related peptide (CGRP). Multiple lines of evidence have implicated CGRP in the pathophysiology of migraine headache making the CGRP receptor an attractive target for development of small-molecule antagonists as a novel treatment for this debilitating condition. The CGRP receptor antagonists telcagepant and olcegepant (BIBN4096BS) have demonstrated clinical efficacy in the treatment of migraine and there is now a need to better understand how these molecules interact with the receptor. Previous work has shown the extracellular portion of RAMP1 to be important for binding of these antagonists, with tryptophan-74 being a key interaction site. The crystal structure of the extracellular portion of human RAMP1 placed tryptophan-74 in a hydrophobic patch hypothesized to interact with CGRP receptor ligands and also identified nearby residues that may be important for ligand binding. In this study we explored the role played by these residues of RAMP1 using an alanine replacement strategy. We confirmed a role for tryptophan-74 in antagonist binding and also identified arginine-67 as being important for binding of telcagepant but not compound 3, a close analog of BIBN4096BS. We also identified tryptophan-84 as being critical for both high-affinity binding of the non-peptide antagonists as well as the peptides CGRP and CGRP(8-37). These data for the first time pinpoint a specific RAMP1 residue important for both antagonist and agonist potency and are consistent with the N-terminal domain of RAMP1 forming the binding pocket interface with CLR.     

Routine assay for detection of IgG and IgM antiglobulins in seronegative and seropositive rheumatoid arthritis.

A convenient technique suitable for the routine estimation of IgM and IgG antiglobulins has been devised. The assay involves the binding of antiglobulins (rheumatoid factors) to rabbit immunoglobulin linked to the surface of plastic tubes; the amount of antiglobulin bound is then determined by adding radiolabelled antihuman IgG or IgM. Both antiglobulins were raised in virtually all seropositive rheumatoid arthritics, and 19 out of 22 seronegative patients had raised values for either IgM or IgG rheumatoid factors. The test should prove valuable in diagnosis and the results further emphasize autosensitization to IgG as a dominant immunological characteristic of different forms of rheumatoid arthritis.     

Biocolloid retention in partially saturated soils

Unsaturated soils are considered excellent filters for preventing the transport of pathogenic biocolloids to groundwater, but little is known about the actual mechanisms of biocolloid retention. To obtain a better understanding of these processes, a number of visualization experiments were performed and analyzed.     

Assembly of the secretion pores GspD,Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA

In Gram‐negative bacteria, β‐barrel proteins are integrated into the outer membrane by the β‐barrel assembly machinery, with key components of the machinery being the Omp85 family members BamA and TamA. Recent crystal structures and cryo‐electron microscopy show a diverse set of secretion pores in Gram‐negative bacteria, with α‐helix (Wza and GspD) or β‐strand (CsgG) transmembrane segments in the outer membrane. We developed assays to measure the assembly of three distinct secretion pores that mediate protein (GspD), curli fibre (CsgG) and capsular polysaccharide (Wza) secretion by bacteria and show that depletion of BamA and TamA does not diminish the assembly of Wza, GspD or CsgG. Like the well characterised pilotins for GspD and other secretins, small periplasmic proteins enhance the assembly of the CsgG β‐barrel. We discuss a model for integral protein assembly into the bacterial outer membrane, focusing on the commonalities and differences in the assembly of Wza, GspD and CsgG.     

Localization of norovirus and poliovirus in Pacific oysters

Aims:  To examine the uptake and tissue distribution of norovirus (NoV) and poliovirus (PV) experimentally bioaccumulated in feeding Pacific oysters ( Crassostrea gigas ).
Methods and Results:  Pacific oysters were allowed to bioaccumulated either PV or NoV under tidally synchronized feeding conditions in laboratory tanks. Oysters were then either fixed and paraffin wax embedded prior to localizing virus within tissues by immunohistochemistry (IHC), or they were dissected into digestive tract (stomach, intestine and digestive diverticula), gill and labial palp tissues, and the viral load determined by quantitative RT-PCR. Both PV and NoV immunoreactivities were predominantly found in the lumen and within cells of the digestive tract tissues; however, PV was also found within cells of nondigestive tract tissues, and in the gills and labial palp. Quantitative RT-PCR of tissue extracts corroborate the immunohistochemical data in that the major site for virus localization is the gut, but significant amounts of viral RNA were identified in the gills and labial palp.
Conclusions:  The human enteric viruses, PV and NoV, are readily bioaccumulated by feeding Pacific oysters and that some of the virus is internalized within cells of both digestive and nondigestive tissues.
Significance and Impact of the Study:  Oysters that have been virally contaminated even after depuration (cleaning) in uncontaminated seawater could pose a human health risk if consumed.     

Simple high-performance liquid chromatographic method for the determination of tocotrienols in human plasma

A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of vitamin E especially δ-, γ- and α-tocotrienols in human plasma. The method entailed direct injection of plasma sample after deproteinization using a 3:2 mixture of acetonitrile–tetrahydrofuran. The mobile phase comprised 0.5% (v/v) of distilled water in methanol. Analyses were run at a flow-rate of 1.5 ml/min with the detector operating at an excitation wavelength of 296 nm and emission wavelength of 330 nm. This method is specific and sensitive, with a quantification limit of approximately 40, 34 and 16 ng/ml for α-, γ- and δ-tocotrienol, respectively. The mean absolute recovery values were about 98% while the within-day and between-day relative standard deviation and percent error values of the assay method were all less than 12.0% for α-, γ- and δ-tocotrienol. The calibration curve was linear over a concentration range of 40–2500, 30–4000 and 16–1000 ng/ml for α-, γ- and δ-tocotrienol, respectively. Application of the method in a bioavailability study for determination of the above compounds was also demonstrated.