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Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the α β δ ε ζ and θ isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH3somatomammotrophic cell line. AtT-20/D16-V corticotrophic and α T3-1 gonadotrophic murine cell lines contained no PKC-δ. The γ or η isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca2+-independent PKCs in αT3-1 and GH3cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca2+-dependent PKC activities were of similar sensitivity to PDBu in GH3, αT3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca2+-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca2+-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)

Structural microdomains of G protein-coupled receptors (GPCRs) consist of spatially related side chains that mediate discrete functions. The conserved helix 2/helix 7 microdomain was identified because the gonadotropin-releasing hormone (GnRH) receptor appears to have interchanged the Asp2.50and Asn7.49residues which are conserved in transmembrane helices 2 and 7 of rhodopsin-like GPCRs. We now demonstrate that different side chains of this microdomain contribute specifically to receptor expression, heterotrimeric G protein-, and small G protein-mediated signaling. An Asn residue is required in position 2.50(87) for expression of the GnRH receptor at the cell surface, most likely through an interaction with the conserved Asn1.50(53)residue, which we also find is required for receptor expression. Most GPCRs require an Asp side chain at either the helix 2 or helix 7 locus of the microdomain for coupling to heterotrimeric G proteins, but the GnRH receptor has transferred the requirement for an acidic residue from helix 2 to 7. However, the presence of Asp at the helix 7 locus precludes small G protein-dependent coupling to phospholipase D. These results implicate specific components of the helix 2/helix 7 microdomain in receptor expression and in determining the ability of the receptor to adopt distinct activated conformations that are optimal for interaction with heterotrimeric and small G proteins.

The characteristics of PKC activation induced by a number of compounds were investigated using PKCs, partially-purified from sources with a naturally high abundance of certain Ca2+ dependent PKC isoforms. Native isoforms were used rather than PKC isoforms expressed from a baculovirus system to assess the effect of tissue specific factors on activity. However, some data using recombinant PKC a were included for comparison.

Certain physiological events in anterior pituitary tissue are regulated by protein kinase C that is relatively resistant to the inhibitor, H7. In the present studies we have shown that two H7-resistant protein kinase C activities may be isolated from (pituitary) gonadotroph-derived αT3-1 cells using hydroxyapatite chromatography. These activities have the characteristics of PKCζ and an apparently novel PKC isoform. The availability of clonal αT3-1 cells will facilitate investigations of the pituitary events.

The depolarisation-induced influx of 45Ca 2+into anterior pituitary tissue and GH 3cells through ‘L’-type, nimodipine-sensitive channels was investigated. In anterior pituitary prisms, phorbol esters, activators of protein kinase C, caused an enhancement of K +-induced 45Ca 2+influx. However, in the GH 3anterior pituitary cell line, phorbol esters inhibited K +-induced 45Ca 2+influx. The modulation by phorbol esters in both tissues was stereo-specific and time- and concentration-dependent. The diacylglycerol analogue, 1,2-dioctanoyl sn-glycerol was able to mimic the phorbol ester-induced enhancement of calcium influx into anterior pituitary pieces, but was ineffective in GH 3cell. 1,2-Diactanoyl sn-glycerol may selectively activate an isoform of protein kinase C which is responsible for enhanced ‘L’-type Ca 2+-channel activity.

Inhibition of phorbol 12,13‐dibutyrate‐induced protein kinase C (PKC) activity from rat midbrain, anterior pituitary and a number of other tissues, as well as COS 7 cells, was studied in vitro. In anterior pituitary, Ca2+‐independent activity was notably resistant to H7 but sensitive to staurosporine and Ro 31‐8220. All Ca2+‐dependent activity was sensitive to these three inhibitors. Mezerein and 1,2‐dioctanoyl‐sn‐glycerol also activated this H7‐insensitive PKC from anterior pituitary. The distribution of this activity, prominently expressed in pituitary and perhaps also lung, and its characteristic resistance to H7 but not other inhibitors, does not obviously correlate with that of any of the well‐characterised PKCs, and may reflect either a novel or a modified isoform.

Western blots of αT3‐1 cell extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. These revealed the presence of PKC types α, ϵ and ζ, but β, γ, δ and η were not detected. The potency with which partially‐purified cytosolic PKC from αT3‐1 cells was activated by phorbol 12,13‐dibutyrate (PDBu), mezerein and 1,2‐dioctanoyl‐sn‐glycerol was assessed in the presence and absence of Ca2+The inhibitors staurosporine, K252a, H7, GF109203X and Ro 31‐8220 were tested on basal activity, PDBu‐induced activity and Ca2++ PDBu‐induced kinase activity. Each inhibitor showed distinct differences in their IC50values under the three conditions, suggesting that these inhibitors may exhibit different potencies on the PKC isoforms present in αT3‐1 cells. Although histone IIIs was used as the phosphate acceptor for most of these experiments, the efficiency of α, ϵ and ζ, peptide and GS peptide substrates were also determined, with ϵ peptide giving the greatest activity in the presence of PDBu or Ca2+. Each substrate displayed a different pattern of activation under the conditions tested. Overall, the findings suggest that 3 or more PKC isoforms with varying specificities are present in gonadotroph‐derived αT3‐1 cells and that the contribution of each isofonn should be considered when these cells are used in models of anterior pituitary cell function where PKC is involved.

The depolarisation‐induced influx of 45Ca2+into anterior pituitary tissue and GH3cells through ‘L’‐type, nimodipine‐sensitive channels was investigated. In anterior pituitary prisms, phorbol esters, activators of protein kinase C, caused an enhancement of K+‐induced 45Ca2+influx. However, in the GH3anterior pituitary cell line, phorbol esters inhibited K+‐induced 45Ca2+influx. The modulation by phorbol esters in both tissues was stereo‐specific and time‐ and concentration‐dependent. The diacylglycerol analogue, 1,2‐dioctanoyl sn‐glycerol was able to mimic the phorbol ester‐induced enhancement of calcium influx into anterior pituitary pieces, but was ineffective in GH3cell. 1,2‐Diactanoyl sn‐glycerol may selectively activate an isoform of protein kinase C which is responsible for enhanced ‘L’‐type Ca2+‐channel activity.

The effect of incubating aT3-1 cells with phorbol 12,13-dibutyrate (PDBu) on the protein kinase C (PKC) isoform content (predominantly a, ? and ? isoforms) was assessed by immunoblotting, enzyme activity assay and [3H]PDBu binding. After exposure to PDBu for 17 h the immunoreactivity detected for both PKC a and PKC ? had disappeared from cytosol and had increased slightly in membranes. Immunoreactivity for PKC ? was present as two bands in cytosol; after PDBu treatment both bands decreased in intensity, the higher molecular weight band more than the lower. The lower molecular weight band corresponded with a component of constitutive PKC activity eluting from DEAE cellulose that was defined by inhibition of basal activity with GF 109203X or H7. Investigation of very short treatment times with PDBu using binding, immunoblot and activity measurements (in the presence/absence of Ca2+) indicated that translocation of PKC a and ? was very rapid — detectable by 10 sec, maximal within minutes. Reduction of these isoforms in membranes took much longer, and was not apparent up to 150 min. The immunoblot data for PKC ? in cytosol showed no detectable effect of PDBu treatment on the low molecular weight band up to 150 min although it was reduced at 17 h. Translocation of the upper band was detectable at 10 sec but this band may have resulted from cross-reaction with other PKC isoforms. The constitutive activity and low molecular weight (‘authentic’) PKC ? immunoreactivity were partially affected after long exposure only, suggesting an action of PDBu on PKC ? secondary to activation of the other PKC isoforms. An endogenous receptor agonist, luteinising hormone-releasing hormone (LHRH), was also used to assess by immunoblotting, translocation of the PKC isoforms. Although all the isoforms did translocate from cytosol to membrane fractions, they did so with distinctly different time courses: PKC ? moved more rapidly than PKC ? which appeared to translocate more quickly than PKC a. After downregulation of the responsive PKC isoforms with PDBu, the remaining PKC ? was not translocated by LHRH. (Mol Cell Biochem 165: 65–75, 1996)

The article discusses the way the Richard Tarling won the Historic final using Jamun T2 overtaking Ben Mitchell who used Merlyn Mk20 and it also highlights the way Northern Pre-90 champion Jaap Blijleven won the non-historic consolation race for Pre-93 cars held in England on November 8, 2017.

The article reports that racer Will Blackwell-Chambers has won the MX-5 championship during the 750 Motor Club season finale in England in 2016 while rival Jack Sycamore has finished in the 36th place for race two due to suspension problems.

The article reports on the performance of automobile race drivers including Paul Smith who won the Formula Vee title, Andrew Thorpe, and Rod Goodman.

The article reports that car racer Robin Gearing has won the 750 Formula title Formula title during 750 Motor Club car racing; but racer Bill Cowley impressed by his return to the series after his accident at Mallory Park.

The article offers information on winning the Formula Vee racing by racing driver Adam Macaulay, winning the Mazda MX-5 racing by racing driver Michael Comber, and winning the Classic Stock Hatch race by racing driver Matt Rozier.

The article presents the results and highlights of the 750 Motor Club car racing tournament held at Donington Park in Leicestershire, England on March 19-20, 2016. The Club Enduro series was won by drivers Ben Demetriou and Jonathan Evans. Also cited are the victory of Ian Allee in race one of the Locost Championship, the win by Adam Shepherd in the M3 Cup, and the double victories by Paul Smith in the Formula Vee race.

BackgroundPhelan and Link argue that socioeconomic status is a fundamental cause of mortality inequalities, hypothesising that any fundamental cause (other candidates include racism and stigma) will have three essential features: (1) will be associated with many disease outcomes; (2) will affect outcomes through a range of intervening mediators; and (3) will persistently display health gradients despite substitution of new for old mediators, such as persistent gradient in all-cause mortality despite change from infectious to non-communicable disease risk factors. They further propose that fundamental causes probably operate via differential access to many resources, which can be variously used to protect and improve health, and that there should therefore be no or lesser health gradients when deployment of resources for health advantage cannot be consciously used; for example, in cases in which we know little or nothing about how to prevent or treat a fatal disease. In view of the already substantial evidence that socioeconomic status meets the first and second essential feature of a fundamental cause as defined by Phelan and Link, we set out to assess whether there is evidence of substitution in a Scottish population sample and investigate whether socioeconomic inequalities in mortality increase with increasing preventability in Scotland.