Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity

The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin's ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues.A.J.R., B.E.F. and M.C.L. acknowledge funding from National Health and Medical Research Council (Project Grant APP1069328 and Project Grant APP1058233) and Australian Research Council. Te Walter and Eliza Hall Institute of Medical Research acknowledges Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS

Insulin in motion: The A6-A11 disulfide bond allosterically modulates structural transitions required for insulin activity

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.The structural transitions required for insulin to activate its receptor and initiate regulation of glucose homeostasis are only partly understood. Here, using ring-closing metathesis, we substitute the A6-A11 disulfide bond of insulin with a rigid, non-reducible dicarba linkage, yielding two distinct stereo-isomers (cis and trans). Remarkably, only the cis isomer displays full insulin potency, rapidly lowering blood glucose in mice (even under insulin-resistant conditions). It also posseses reduced mitogenic activity in vitro. Further biophysical, crystallographic and molecular-dynamics analyses reveal that the A6-A11 bond configuration directly affects the conformational flexibility of insulin A-chain N-terminal helix, dictating insulin’s ability to engage its receptor. We reveal that in native insulin, contraction of the Cα-Cα distance of the flexible A6-A11 cystine allows the A-chain N-terminal helix to unwind to a conformation that allows receptor engagement. This motion is also permitted in the cis isomer, with its shorter Cα-Cα distance, but prevented in the extended trans analogue. These findings thus illuminate for the first time the allosteric role of the A6-A11 bond in mediating the transition of the hormone to an active conformation, significantly advancing our understanding of insulin action and opening up new avenues for the design of improved therapeutic analogues

Cause of Death and Predictors of All-Cause Mortality in Anticoagulated Patients With Nonvalvular Atrial Fibrillation : Data From ROCKET AF

M. Kaste on työryhmän ROCKET AF Steering Comm jäsen.Background-Atrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all-cause mortality may guide interventions. Methods and Results-In the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all-cause mortality in the 14 171 participants in the intention-to-treat population. The median age was 73 years, and the mean CHADS(2) score was 3.5. Over 1.9 years of median follow-up, 1214 (8.6%) patients died. Kaplan-Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all-cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33-1.70, P= 75 years (hazard ratio 1.69, 95% CI 1.51-1.90, P Conclusions-In a large population of patients anticoagulated for nonvalvular atrial fibrillation, approximate to 7 in 10 deaths were cardiovascular, whereasPeer reviewe

Management of Cutaneous Drug Reactions

Drugs are potent chemicals that often have effects in the body beyond the desired action. These effects may range from mild and expected side effects to dramatic and life-threatening anaphylaxis. Adverse drug reactions account for between 2% and 6% of hospital admissions and may prevent administration of otherwise effective therapeutic agents. Cutaneous and mucocutaneous eruptions are the most common adverse reactions to oral or parenteral drug therapy, and the spectrum ranges from transitory exanthematous rash to the potentially fatal toxic epidermal necrolysis. Different mechanisms, including both immunologic and nonimmunologic, are responsible for cutaneous adverse drug reaction. The treatment of cutaneous drug eruptions essentially rests on accurate history, a thorough physical examination, discontinuation of the offending drug, and supportive care. The management of a cutaneous drug eruption is very much individualized, based on the clinical setting. This review aims to provide a general approach to the patient with a presumed cutaneous drug reaction

Traditional therapies: glucocorticoids, azathioprine, methotrexate, hydroxyurea

The 'old favourites' used for treatment of inflammatory diseases, and hence, the original immunomodulators, include the glucocorticoids, azathioprine, methotrexate and hydroxyurea. Glucocorticoids are still one of the most effective anti-inflammatory agents because they work on several different intracellular processes and hence, block many components that contribute to inflammatory and immune responses. They bind to intracellular glucocorticoid receptors which transport them into the nucleus. Here the receptor/steroid complex may bind to many genes that interact with transcription factors including NF?B and AP-1, to inhibit their activation, thereby preventing activation of many genes encoding immune effector and pro-inflammatory cytokines. Also, protein kinases involved in intracellular signalling, are directly activated resulting in phosphorylation of various targets of which Annexin (AXA)-1 is critical in inhibiting biosynthesis of both purines and DNA. This results in reduced proliferation of B and T lymphocytes, reduced immune effector mechanisms and reduced recruitment of mononuclear cells including monocytes into sites of immune inflammation. Methotrexate also blocks DNA synthesis and hence cellular proliferation but also induces release of adenosine. This inhibits chemotaxis of polymorph neutrophils and release of critical cytokines such as TNF-? and Interleukins 6 and 8. Hydroxyurea also inhibits DNA synthesis with inhibitory effects on proliferation of lymphocytes and possibly kerationcytes.Even though many new agents with much greater selectivity are coming through into clinical use, this group of old agents still have an absolutely central position in the therapeutic armamentarium. Their value lies in the fact that they are not 'clean' drugs with narrow effects but they inhibit a wide range of mechanisms involved in immune and inflammatory processes

Ultraviolet-B-induced erythema is mediated by nitric oxide and prostaglandin E2 in combination

Ultraviolet-B-induced erythema (one, two, or four times the minimal erythema dose) was reduced but not abolished by application of 1% indomethacin gel immediately after irradiation of human skin. Continuous synthesis of prostaglandins is reflected by similar levels of indomethacin-mediated inhibition of erythema at any time within 48 h after irradiation. Repeated applications of indomethacin did not increase the inhibition. Twenty-four hours after irradiation with four minimal erythema doses, mean prostaglandin E2 levels in suction blisters were 27.2 ng per ml (SEM 11) compared with 8.6 ng per ml in unirradiated skin (n = 25; p < 0.01). Prosta glandin E2 levels in dermal tissues, sampled by microdialysis (depth 0.6 +/- 0.1 mm), were 310 pg per ml (SEM 123) and 237 pg per ml (SEM 88) in irradiated and unirradiated skin, respectively (n = 7, n.s.). Nitric oxide also made a significant contribution to ultraviolet-B-induced erythema. Ultraviolet erythema was inhibited by L-NAME in a dose-related fashion with 2 mM L-NAME causing total abolition of the response. L-NAME was effective at all time points up to 48 h suggesting that NO was produced continuously. NO was undetectable in suction blister fluid but in dermal microdialysate NO was present at 44.3 ng per ml (SEM 6.2) following ultraviolet B compared with 26.0 ng per ml (SEM 8.0) in unirradiated skin (p < 0.05), approximately 1000 times the molar concentration of prostaglandin E2. These findings confirm prostaglandin E2 and NO to be mediators of ultraviolet-induced erythema. They also show that there is prolonged synthesis of both mediators within the erythemal response and that synthesis of NO is induced by lower doses of ultraviolet B compared with that of prostaglandin E2.Abbreviations: COX, cyclo-oxygenase; NOS, nitric oxide synthas

In vitro diagnostic assays are effective during the acute phase of delayed-type drug hypersensitivity reactions

BackgroundPrevious reports have suggested that drug-specific lymphocyte proliferation assays (LPA) can be used retrospectively to confirm the culprit drug following delayed-type drug hypersensitivity reactions (DHR). However, only limited evidence supports their use in aiding acute clinical management. The aim of this study was to compare the LPA against combination cytokine assays for potential use in the acute setting.MethodsA total of 43 patients with DHR (19 during the acute reaction, 20 after recovery, four during acute and after recovery) and 14 control subjects without DHR were investigated using ex vivo analysis of drug-specific proliferation, and interferon (IFN)-? and interleukin (IL)-4 production.ResultsHealthy controls showed negative drug-specific proliferation and cytokine release in contrast to individuals with a known sensitivity (P < 0·0001). The assays demonstrated a test specificity of 95% (LPA), 83% (IFN-?) and 92% (IL-4). The sensitivity of combined measurement of drug-specific IFN-? and IL-4 cytokines during acute DHR was better than LPA (82% vs. 50%), but all assays were less sensitive during the recovery phase. The correlation between LPA and IFN-? assays was strong (r = 0·7, P < 0·0001), whereas the IL-4 assay did not correlate as well with either of these assays. In contrast to LPA, drug enzyme-linked immunosorbent spot assays showed positive responses in patients concurrently taking immunosuppressive medication.ConclusionsIn vitro assays of drug-specific IFN-? and IL-4 production offer potential for use as rapid diagnostic tests. Cytokine detection offers distinct advantages over the LPA, including a shorter assay time, a greater sensitivity and effectiveness in testing immunosuppressed patients

Segmental cherry angiomas associated with extragenital lichen sclerosus: a report of two cases

Cherry angiomas (Campbell de Morgan spots) are common acquired red skin papules composed of dilated capillary loops, usually of unknown aetiology. Extragenital lichen sclerosus (LS) presents as porcelain-white scaly atrophic lesions with or without genital involvement. We report two cases of segmental multiple cherry angiomas in association with extragenital LS. Two unrelated women, aged 46 and 66 years, presented with extragenital LS affecting their axillae and lower abdomen. During the examination, both patients were noted to have several hundred red skin papules in a segmental distribution, affecting the left thigh and flank of one woman, and the right abdomen and back of the other. Clinically and histologically, the papules were consistent with cherry angiomas. The striking segmental distribution of multiple cherry angiomas may be due to genetic mosaicism; however, segmental Fabry disease was excluded by sequence analysis of the α-galactosidase A gene. Any causal link between cherry angiomas and LS remains uncertain