Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.     

The cloned murine M1 muscarinic receptor is associated with the hydrolysis of phosphatidylinositols in transfected murine B82 cells

A rat genomic DNA clone was isolated by its homology with a conserved primary sequence among the mammalian and avian beta adrenergic and porcine muscarinic receptors. A gene identified in this clone was highly homologous to the rat M1 muscarinic receptor. Stable expression of this gene was achieved in an established murine fibroblast cell line, B82. The gene product exhibits M1 type muscarinic receptor characteristics, as it has high affinity for PZ but low affinity for AF-DX 116. Carbachol stimulated the hydrolysis of phosphatidylinositols in the transfected cells. Pirenzepine had a more potent inhibitory effect on this response than AF-DX 116 since their functional inhibition constants were 13 nM and 480 nM, respectively, which is consistent with an M1 pharmacological profile. These data suggest that the M1 muscarinic receptor encoded by the gene is coupled to the hydrolysis of phosphatidylinositols after transfecting this gene into the B82 cells.     

News from the Biological Stain Commission No. 5

In this fifth issue of News from the Biological Stain Commission (BSC), under the heading of Regulatory Affairs, the BSC's International Affairs Committee provides more information from the meeting of the International Standards Organization ISO/TC 212 Committee that took place on June 2–4, 2008 at Vancouver, Canada. In addition, we give an update on the current situation regarding the supplies of hematoxylin.     

Benzidine-based dyes: effects of industrial practices,regulations, and world trade on the biological stains market

One of the most sweeping changes in the dye industry since the advent of synthetic dyes grew out of the health risks associated with benzidine. Dyes made from benzidine and its derivatives were used around the world until adverse health effects become incontrovertible. Workers and family members of workers involved in production and use of benzidine-based dyes had a high incidence of bladder cancer. Following publication of several reports documenting this health hazard, dye makers in the USA, Europe, and Japan phased these dyes out of production in the 1970s. Government regulations lent legal support for these voluntary initiatives. Two strategies subsequently evolved to compensate: developed nations brought alternative substances to market while emerging countries increased production of carcinogenic dyes and sold them at discount prices around the world. Nearly all dye manufacturing now has moved away from nations whose costs of production and compliance rendered them unable to compete. The purpose of this brief review is to publicize the health risks associated with dyes made from benzidine and its congeners, and to alert all companies and end users handling these dyes for biomedical applications that composition of the product and lot-to-lot variability may be problematic because of the manufacturing and distribution practices of the countries where they are produced.     

Dye–tissue interactions: mechanisms,quantification and bonding parameters for dyes used in biological staining

Staining of tissues by dyes is accomplished through various types of bonds, some of which have been poorly defined in traditional biological literature. Here, basic principles of bonding are reviewed to establish uniform terminology and definitions consistent with the field of chemistry. The concept of charge – its presence or absence, magnitude, extent of delocalization and potential for being displaced by outside forces – underlies all bonding phenomena. These same attributes influence solubility and resistance to extraction during dehydration of tissue sections. Covalent bonds involve shared electrons; they are very strong and essentially irreversible under conditions encountered during staining. Polar covalent bonds within dye molecules generate partial atomic charges that create the potential for hydrogen bonding. This is measured by the hydrogen bonding parameter (h), the number of groups bearing charges within the ranges ?0.15 to ?0.50 eV or +0.15 to +0.30 eV. The potential for ionic bonding is indicated by net charge (Z), while the strength of such bonds is a function of charge site geometry on both bonding partners. Charge delocalization owing to conjugation, electron influencing groups, and resonance creates soft charge sites in which the ionic charge is spread over a large volume. Poorly delocalized charges or point charges are hard (small in volume). Firm bonds result from hard-hard or soft-soft pairs. Hard-soft combinations are weak, readily displaced in competitive interactions, and disrupted by solvents. Coordinate bonds with certain metals are involved with mordant staining and metal chelation dyes. Three different van der Waals attractions comprise the remainder of bonding types, all involving dipoles: Keesom (dipole-dipole) forces, Debye (dipole-induced dipole) forces and London (induced dipole-induced dipole) forces. Potentials for engaging in any of these is quantified by measures of polarity (dipole moment, d), polarizability (crudely with π atoms describing the size of the conjugated system, or more directly with α), hydrophobicity (with the octanol-water partition coefficient, log P or the more convenient Hydrophobic Index, HI), and the number of halogen atoms (X). By using molecular modeling software, quantitative measures of bonding potential (bonding parameters) have been determined for over 400 dyes.     

Effects of fixation on routine,special and immunohistochemical stains: introduction to a symposium for the Biological Stain Commission

Orcein was first proposed as an elastic fiber stain by Taenzer in 1890, and has proved very useful for the purpose. It is an important constituent of the author's “Quad” stain. Unfortunately not all orcein samples have proved equally satisfactory, whether they are derived from the lichens from which the dye was originally prepared or have been manufactured by a synthetic process. At the present time several brands are available, and two of the brands of the synthetic product investigated have not proved to be the same thing; one of the latter proves only fair as an elastin stain, the other is one of the best samples the author has ever tried.     

A cross-sectional study to determine the seroprevalence of bluetongue virus serotype 8 in sheep and goats in 2006 and 2007 in the Netherlands

Background

In August 2006 a major epidemic of bluetongue virus serotype 8 (BTV8) started off in North-West Europe. In the course of 2007 it became evident that BTV8 had survived the winter in North-West Europe, re-emerged and spread exponentially. Recently, the European Union decided to start vaccination against BTV8. In order to improve the understanding of the epidemiological situation, it was necessary to execute a cross-sectional serological study at the end of the BT vector season. Cattle were the target species for cross-sectional serological studies in Europe at the end of 2006 and 2007. However, there was no information on the BTV8-seroprevalence in sheep and goats.

Results

On the basis of our cross-sectional study, the estimated seroprevalence of BTV8-exposed locations in the Netherlands in 2006 was 0% for goats (95% confidence interval: 0 – 5.6%) and 7.0% for sheep (95% confidence interval: 3.5 – 12.9%). The estimated seroprevalence of BTV-8 exposed locations in 2007 was 47% for goats (95% confidence interval: 36 – 58%) and 70% for sheep (95% confidence interval: 63 – 76%). There was a wide range in within-location seroprevalence in locations with goats and sheep (1 – 100%). A gradient in seroprevalence was seen, with the highest level of seroprevalence in the southern Netherlands, the area where the epidemic started in 2006, and a decreasing seroprevalence when going in a northern direction.

Conclusion

There is a much higher estimated seroprevalence of locations with goats exposed to BTV8 than can be inferred from the rather low number of reported clinical outbreaks in goats. This is probably due to the fact that clinical signs in infected goats are far less obvious than in sheep. The wide range in within-location seroprevalence observed means that the proportion of animals protected in 2008 by a natural infection in 2006 and/or 2007 can differ highly between flocks. This should be taken into account when vaccinating animals.

     

Local spread of classical swine fever upon virus introduction into The Netherlands: Mapping of areas at high risk

Background

In the recent past, the introduction of Classical Swine Fever Virus (CSFV) followed by between-herd spread has given rise to a number of large epidemics in The Netherlands and Belgium. Both these countries are pork-exporting countries. Particularly important in these epidemics has been the occurrence of substantial "neighborhood transmission" from herd to herd in the presence of base-line control measures prescribed by EU legislation. Here we propose a calculation procedure to map out "high-risk areas" for local between-herd spread of CSFV as a tool to support decision making on prevention and control of CSFV outbreaks. In this procedure the identification of such areas is based on an estimated inter-herd distance dependent probability of neighborhood transmission or "local transmission". Using this distance-dependent probability, we derive a threshold value for the local density of herds. In areas with local herd density above threshold, local transmission alone can already lead to epidemic spread, whereas in below-threshold areas this is not the case. The first type of area is termed 'high-risk' for spread of CSFV, while the latter type is termed 'low-risk'.

Results

As we show for the case of The Netherlands, once the distance-dependent probability of local transmission has been estimated from CSFV outbreak data, it is possible to produce a map of the country in which areas of high-risk herds and of low-risk herds are identified. We made these maps even more informative by estimating border zones between the two types of areas. In these border zones the risk of local transmission of infection to a nearby high-risk area exceeds a certain level.

Conclusion

The risk maps provide an easily understandable visualization of the spatial heterogeneities in transmission risk. They serve as a tool for area-specific designs of control strategies, and possibly also for spatial planning of areas where livestock farming is allowed. Similar risk maps can in principle be constructed for other highly-transmissible livestock infections that spread via neighborhood transmission.

     

Artificial ion channels formed by a synthetic cyclic peptide.

A new cyclic peptide 1 having an (LLLD)3 configuration pattern was designed that is capable of forming artificial transmembrane ion channels by self-assembly of planar peptide rings, with hydrophilic groups arrayed in the interior of the channel. Ion permeability in the presence of the synthetic peptide 1, cyclo[-Trp-Dap-Leu-D-Ala-Trp-Ser-Val-D-Ala-Trp-Ser-Ile-Gly-] (Dap: L-diaminopropionic acid), was observed in lipid bilayer membranes. The pH dependence of ionic conductance showed that the beta-amino group of Dap may play a role in the conductance of the peptide channels. Fourier-transform infrared and circular dichroism data imply that, in a membrane, a stack of cyclic peptides is formed in which the inter peptide H bonds form a kind of beta-structure analogous to that in the gramicidin A dimer and distinct from the H-bonding pattern of the beta-barrels.