Corticostatic peptides.

In the last four years corticostatic (anti-ACTH) peptides have been isolated from human, rabbit, guinea pig and rat tissues. These peptides do not act via the cAMP cell signalling system but rather via the inhibition of the binding of ACTH to its receptor most probably through direct competition with the 14-18 sequence of ACTH for receptor binding. ACTH has specific high affinity receptors on adrenal cells but rabbit corticostatin I (CSI) has high capacity, low affinity receptors which are competed for by unlabelled excess CSI but not by excess ACTH. This indicates the presence of specific CSI adrenal cell receptors. The rabbit pituitary, hypothalamus, thalamus, adrenals, lungs and placenta contain sizeable amounts of immunoassayable CSI. Immunochemical localization of CSI indicates that it is present in the large macrophages and in neutrophils in rabbit lung, in macrophages and "supporting" endothelial cells in the spleen and in the adrenals in the cells of the zona reticularis. We have also isolated and identified new peptides which contain 12 cysteines from immune cells of humans, rats and a teleost, the carp. The functions of these peptides are now being determined. This large family of peptides may have many other, yet unidentified functions but at present we can only describe a small number of these.     

Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling,cross-linking,and mutagenesis

The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits.     

Beta 2-adrenergic receptor stimulated,G protein-coupled receptor kinase 2 mediated,phosphorylation of ribosomal protein P2

G protein-coupled receptor kinases are well characterized for their ability to phosphorylate and desensitize G protein-coupled receptors (GPCRs). In addition to phosphorylating the beta2-adrenergic receptor (beta2AR) and other receptors, G protein-coupled receptor kinase 2 (GRK2) can also phosphorylate tubulin, a nonreceptor substrate. To identify novel nonreceptor substrates of GRK2, we used two-dimensional gel electrophoresis to find cellular proteins that were phosphorylated upon agonist-stimulation of the beta2AR in a GRK2-dependent manner. The ribosomal protein P2 was identified as an endogenous HEK-293 cell protein whose phosphorylation was increased following agonist stimulation of the beta2AR under conditions where tyrosine kinases, PKC and PKA, were inhibited. P2 along with its other family members, P0 and P1, constitutes a part of the elongation factor-binding site connected to the GTPase center in the 60S ribosomal subunit. Phosphorylation of P2 is known to regulate protein synthesis in vitro. Further, P2 and P1 are shown to be good in vitro substrates for GRK2 with K(M) values approximating 1 microM. The phosphorylation sites in GRK2-phosphorylated P2 are identified (S102 and S105) and are identical to the sites known to regulate P2 activity. When the 60S subunit deprived of endogenous P1 and P2 is reconstituted with GRK2-phosphorylated P2 and unphosphorylated P1, translational activity is greatly enhanced. These findings suggest a previously unrecognized relationship between GPCR activation and the translational control of gene expression mediated by GRK2 activation and P2 phosphorylation and represent a potential novel signaling pathway responsible for P2 phosphorylation in mammals.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.     

Sex differences in cannabinoid pharmacology: A reflection of differences in the endocannabinoid system?

Marijuana is the most widely used illicit drug in the U.S., and marijuana use by women is on the rise. Women have been found to be more susceptible to the development of cannabinoid abuse and dependence, have more severe withdrawal symptoms, and are more likely to relapse than men. The majority of research in humans suggests that women are more likely to be affected by cannabinoids than men, with reports of enhanced and decreased performance on various tasks. In rodents, females are more sensitive than males to effects of cannabinoids on tests of antinociception, motor activity, and reinforcing efficacy. Studies on effects of cannabinoid exposure during adolescence in both humans and rodents suggest that female adolescents are more likely than male adolescents to be deleteriously affected by cannabinoids. Sex differences in response to cannabinoids appear to be due to activational and perhaps organizational effects of gonadal hormones, with estradiol identified as the hormone that contributes most to the sexually dimorphic effects of cannabinoids in adults. Many, but not all sexually dimorphic effects of exogenous cannabinoids can be attributed to a sexually dimorphic endocannabinoid system in rodents, although the same has not yet been established firmly for humans. A greater understanding of the mechanisms underlying sexually dimorphic effects of cannabinoids will facilitate development of sex-specific approaches to treat marijuana dependence and to use cannabinoid-based medications therapeutically.     

BACE-1 inhibitors part 3: identification of hydroxy ethylamines (HEAs) with nanomolar potency in cells

This article is focusing on further optimization of previously described hydroxy ethylamine (HEA) BACE-1 inhibitors obtained from a focused library with the support of X-ray crystallography. Optimization of the non-prime side of our inhibitors and introduction of a 6-membered sultam substituent binding to Asn-294 as well as a fluorine in the C-2 position led to derivatives with nanomolar potency in cell-based assays.     

Synthesis of a [6-pyridinyl-18F]-labelled fluoro derivative of WAY-100635 as a candidate radioligand for brain 5-HT1A receptor imaging with PET

In recent years, considerable effort has been spent on the design, synthesis and pharmacological characterization of radiofluorinated derivatives of the 5-HT(1A) receptor antagonist, WAY-100635, for the in vivo study of these receptors in human brain with PET. (Pyridinyl-6)-fluoro- and (pyridinyl-5)-fluoro-analogues of WAY-100635 (6-fluoro and 5-fluoro-WAY-100635, 5a/6a) were synthesized as well as the corresponding chloro-, bromo- and nitro-derivatives as precursors for labelling (5b-d and 6b-d). Comparative radiolabelling of these precursors with fluorine-18 (positron-emitting isotope, 109.8 min half-life) clearly demonstrated that only ortho-fluorination in this pyridine series, and not meta-fluorination, is of interest for the preparation of a radioligand by nucleophilic heteroaromatic substitution. 6-[(18)F]Fluoro-WAY-100635 ([(18)F]5a) can be efficiently synthesized in one step, either from the corresponding 6-bromo precursor (using conventional heating at 145 degrees C for 10 min) or from the corresponding 6-nitro precursor (using microwave activation at 100 W for 1 min). Typically, 15-25 mCi (0.55-0.92 GBq) of 6-[(18)F]fluoro-WAY-100635 ([(18)F]5a, 1-2 Ci/micromol or 37-72 GBq/micromol) were obtained in 50-70 min starting from a 100 mCi (3.7 GBq) aliquot of a batch of cyclotron-produced [(18)F]fluoride. This (18)F-labelled radioligand is now being evaluated in PET studies.     

Towards the elucidation of the true impact of adipocytokines on cardiovascular risk in rheumatoid arthritis

Adipo(cyto)kines are mostly produced by adipose tissue and orchestrate the adverse impact of excess adiposity on cardiovascular risk. Adipokines also contribute importantly to the pathophysiology of rheumatoid arthritis. Congruent with data reported in previous investigations, Kang and colleagues report in this issue of Arthritis Research & Therapy that adipokine concentrations are further associated with metabolic risk and inflammation and that the leptin–adiponectin ratio associates with the carotid artery resistive index in rheumatoid arthritis. Guided by evidence reported thus far on cardiovascular risk, we discuss six reasons why careful elucidation of adipokine–cardiovascular risk relations is needed in rheumatoid arthritis.In this issue of Arthritis Research & Therapy, Kang and colleagues investigate whether adipokines could link inflammation, metabolic risk factors and cardiovascular disease in rheumatoid arthritis (RA) [1]. Evidence in support of this paradigm was reported previously [2-6]. Patients with RA experience a markedly increased cardiovascular risk that is driven by metabolic risk factors and by high-grade inflammation [7]. Kang and colleagues measured adiponectin, leptin, resistin, tumor necrosis factor alpha and interleukin-6 concentrations and assessed the common carotid artery intima-media thickness, resistive index (RI) and plaque presence by high-resolution ultrasonography [1]. Concentrations of some of the adipokines related to inflammatory markers including C-reactive protein levels and the erythrocyte sedimentation rate, and to metabolic syndrome features.In a previous study by our group, leptin and adiponectin concentrations were not associated with carotid intima-media thickness and plaque [3]. In addition, the leptin–adiponectin ratio and carotid RI as markers of cardiovascular risk have not been reported in RA. For these reasons, besides the abovementioned analyses, Kang and colleagues assessed (only) the relationship of the leptin–adiponectin ratio with carotid RI. In univariate analysis, the leptin–adiponectin ratio as well as age, homeostasis model assessment for insulin resistance, waist circumference and body mass index were associated with the carotid RI. Importantly, in multivariate analysis, only age and the leptin–adiponectin ratio remained significantly related to the carotid RI. The leptin–adiponectin ratio may thus provide information about the presence of subclinical cardiovascular disease beyond that on insulin resistance as assessed by the homeostasis model of insulin resistance, as well as adiposity extent as represented by body mass index and waist circumference in RA.Adipo(cyto)kines comprise a vast range of disparate soluble bioactive proteins that are mostly secreted by adipose tissue [8]. These molecules participate in biological processes that include inflammatory responses and thereby orchestrate the adverse impact of excess adiposity on cardiovascular risk and incident type 2 diabetes [8]. Adipokines represent both adiposity extent and biological activity. RA is a prototypic inflammatory disease. In this context, ~200 recently reported investigations substantiate an important involvement of adipokines in RA activity and severity [9]. By contrast, despite the contribution of adipokines to altered cardiovascular risk in non-RA subjects and the enhanced cardiovascular risk in RA, there is a striking paucity of reported studies on the potential role of adipokines in atherogenesis in RA.A myriad of pertinent reasons exist why the role of adipokines in cardiovascular risk amongst patients with RA requires thorough elucidation. First, RA can modify adipokine production [3,9].Second, and presumably more important, the presence of autoimmunity can alter the effects of adipokines on cardiovascular risk [3,4]. In non-RA subjects, adiponectin production decreases with increasing adiposity and this adipokine has anti-inflammatory properties [8]. However, in RA adiponectin has marked proinflammatory properties [9]. In fact, in Kang and colleagues’ study the adiponectin concentrations were paradoxically positively associated with the erythrocyte sedimentation rate [1]. Whereas in non-RA subjects adiponectin improves metabolic risk and also directly inhibits atherogenesis, we reported recently that in RA, upon using comprehensive potential confounder-adjusted analysis, adiponectin concentrations associated paradoxically with high blood pressure [3,4] and in white but not black Africans with enhanced endothelial activation [4]. Endothelial activation mediates the very initial stages of atherosclerosis [3-6]. Whether such paradoxical relations represent altered effects mediated by RA or a compensatory increase in adiponectin production in the presence of heightened cardiovascular risk and in an attempt to reduce this risk needs further investigation [4].Third, conventional risk factors and disease characteristics can impact on adipokine–atherogenesis relationships in RA [5]. Resistin concentrations thus associate independently with endothelial activation in RA, but this relation is present only in those with, and not in those without, traditional risk factors, abdominal obesity, joint damage as reflected by the presence of deformed joints or prolonged disease duration [5]. This observation further supports the need for sensitivity analysis in the present context. By contrast, interleukin-6 concentrations are more consistently associated with endothelial activation in RA [6].Fourth, the effects of adipokines on cardiovascular risk require examination prior to targeting the respective molecules in an attempt to reduce disease activity and severity in RA [3]. Indeed, should the protective effect of adiponectin on cardiovascular risk be preserved amongst patients with RA, then its blockade would be expected to further enhance cardiovascular risk [3].Fifth, RA influences adiposity and its distribution, which also associates with atherosclerosis in this disease [7,10].Finally, as illustrated by the disparity in adiponectin–endothelial activation relations amongst Africans previously alluded to, population origin impacts on adipokine–cardiovascular risk relations in RA [4].A caveat of Kang and colleagues’ study is that potential confounders were not systematically identified. For example, gender, cardiovascular drug use, antirheumatic agent use and the glomerular filtration rate can all influence both the concentrations and effects of adipokines [3-6]. Nevertheless, this investigation reinforces previously reported evidence that strongly suggests an intriguing and important involvement of adipokines in RA atherogenesis.