Enzymatic Characterization of a Human Acyltransferase Activity

Non-histone protein acylation is increasingly recognized as an important posttranslational modification, but little is known as to the biochemical properties of protein serine acylating enzymes.We here report that we have identified a metal-stimulated serine octanoyltransferase activity in microsomes from human erythroleukemic (HEL) cells. The HEL acylating enzyme was linear with respect to time and protein, exhibited a neutral pH optimum (stimulated by cobalt and zinc), and inhibited by chelating reagents. Hydroxylamine treatment removed most, but not all, of the attached radioactivity. A salt extract of microsomal membranes contained the major portion of enzyme activity, indicating that this acyltransferase is not an integral membrane protein. Sucrose density fractionation showed that the acyltransferase activity is concentrated in the endoplasmic reticulum. In competition experiments, the acyltransferase was well inhibited by activated forms of fatty acids containing at least eight to fourteen carbons, but not by acetyl CoA. The zinc-stimulated HEL acyltransferase did not octanoylate proenkephalin, proopiomelanocortin, His-tagged proghrelin, or proghrelin lacking the amino-terminal His-tag stub of Gly-Ala-Met. The peptides des-acyl ghrelin and ACTH were also not acylated; however, des-acyl ghrelin containing the N-terminal tripeptide Gly-Ala-Met was acylated. Mutagenesis studies indicated a requirement for serine five residues from the amino terminus, reminiscent of myristoyl transferase, but not of ghrelin acylation. However, recombinant myristoyl transferase could not recapitulate the hydroxylamine sensitivity, zinc-stimulation, nor EDTA inhibition obtained with HEL acyltransferase, properties preserved in the HEL cell enzyme purified through four sequential chromatographic steps.In conclusion, our data demonstrate the presence of a zinc-stimulated acyltransferase activity concentrated in the endoplasmic reticulum in HEL cells which is likely to contribute to medium-chain protein lipidation

Dutch randomized trial comparing standard catheter-directed thrombolysis versus Ultrasound-accElerated Thrombolysis for thromboembolic infrainguinal disease (DUET): design and rationale

Background: The use of thrombolytic therapy in the treatment of thrombosed infrainguinal native arteries and bypass grafts has increased over the years. Main limitation of this treatment modality, however, is the occurrence of bleeding complications. Low intensity ultrasound (US) has been shown to accelerate enzymatic thrombolysis, thereby reducing therapy time. So far, no randomized trials have investigated the application of US-accelerated thrombolysis in the treatment of thrombosed infra-inguinal native arteries or bypass grafts. The DUET study (Dutch randomized trial comparing standard catheter-directed thrombolysis versus Ultrasound-accElerated Thrombolysis for thrombo-embolic infrainguinal disease) is designed to assess whether US-accelerated thrombolysis will reduce therapy time significantly compared with standard catheter-directed thrombolysis.Methods/design: Sixty adult patients with recently (between 1 and 7 weeks) thrombosed infrainguinal native arteries or bypass grafts with acute limb ischemia class I or IIa, according to the Rutherford classification for acute ischemia, will be randomly allocated to either standard thrombolysis (group A) or US-accelerated thrombolysis (group B). Patients will be recruited from 5 teaching hospitals in the Netherlands during a 2-year period. The primary endpoint is the duration of catheter-directed thrombolysis needed for uninterrupted flow in the thrombosed infrainguinal native artery or bypass graft, with outflow through at least 1 crural artery.Discussion: The DUET study is a randomized controlled trial that will provide evidence of whether US-accelerated thrombolysis will significantly reduce therapy time in patients with recently thrombosed infrainguinal native arteries or bypass grafts, without an increase in complications. Trial registration: Current Controlled Trials ISRCTN72676102

Unacylated Ghrelin Rapidly Modulates Lipogenic and Insulin Signaling Pathway Gene Expression in Metabolically Active Tissues of GHSR Deleted Mice

Background: There is increasing evidence that unacylated ghrelin (UAG) improves insulin sensitivity and glucose homeostasis; however, the mechanism for this activity is not fully understood since a UAG receptor has not been discovered. Methodology/Principal Findings: To assess potential mechanisms of UAG action in vivo, we examined rapid effects of UAG on genome-wide expression patterns in fat, muscle and liver of growth hormone secretagogue receptor (GHSR)-ablated mice using microarrays. Expression data were analyzed using Ingenuity Pathways Analysis and Gene Set Enrichment Analysis. Regulation of subsets of these genes was verified by quantitative PCR in an independent experiment. UAG acutely regulated clusters of genes involved in glucose and lipid metabolism in all three tissues, consistent with enhancement of insulin sensitivity. Conclusions/Significance: Fat, muscle and liver are central to the control of lipid and glucose homeostasis. UAG rapidly modulates the expression of metabolically important genes in these tissues in GHSR-deleted mice indicating a direct, GHSRindependent, action of UAG to improve insulin sensitivity and metabolic profile

Ghrelin Modulates the fMRI BOLD Response of Homeostatic and Hedonic Brain Centers Regulating Energy Balance in the Rat

The orexigenic gut-brain peptide, ghrelin and its G-protein coupled receptor, the growth hormone secretagogue receptor 1a (GHS-R1A) are pivotal regulators of hypothalamic feeding centers and reward processing neuronal circuits of the brain. These systems operate in a cooperative manner and receive a wide array of neuronal hormone/transmitter messages and metabolic signals. Functional magnetic resonance imaging was employed in the current study to map BOLD responses to ghrelin in different brain regions with special reference on homeostatic and hedonic regulatory centers of energy balance. Experimental groups involved male, ovariectomized female and ovariectomized estradiol-replaced rats. Putative modulation of ghrelin signaling by endocannabinoids was also studied. Ghrelin-evoked effects were calculated as mean of the BOLD responses 30 minutes after administration. In the male rat, ghrelin evoked a slowly decreasing BOLD response in all studied regions of interest (ROI) within the limbic system. This effect was antagonized by pretreatment with GHS-R1A antagonist JMV2959. The comparison of ghrelin effects in the presence or absence of JMV2959 in individual ROIs revealed significant changes in the prefrontal cortex, nucleus accumbens of the telencephalon, and also within hypothalamic centers like the lateral hypothalamus, ventromedial nucleus, paraventricular nucleus and suprachiasmatic nucleus. In the female rat, the ghrelin effects were almost identical to those observed in males. Ovariectomy and chronic estradiol replacement had no effect on the BOLD response. Inhibition of the endocannabinoid signaling by rimonabant significantly attenuated the response of the nucleus accumbens and septum. In summary, ghrelin can modulate hypothalamic and mesolimbic structures controlling energy balance in both sexes. The endocannabinoid signaling system contributes to the manifestation of ghrelin’s BOLD effect in a region specific manner. In females, the estradiol milieu does not influence the BOLD response to ghrelin

Prolactinomas, Cushing's disease and acromegaly: debating the role of medical therapy for secretory pituitary adenomas

Pituitary adenomas are associated with a variety of clinical manifestations resulting from excessive hormone secretion and tumor mass effects, and require a multidisciplinary management approach. This article discusses the treatment modalities for the management of patients with a prolactinoma, Cushing's disease and acromegaly, and summarizes the options for medical therapy in these patients

Value of risk scores in the decision to palliate patients with ruptured abdominal aortic aneurysm

Background: The aim of this study was to develop a 48-h mortality risk score, which included morphology data, for patients with ruptured abdominal aortic aneurysm presenting to an emergency department, and to assess its predictive accuracy and clinical effectiveness in triaging patients to immediate aneurysm repair, transfer or palliative care. Methods: Data from patients in the IMPROVE (Immediate Management of the Patient With Ruptured Aneurysm: Open Versus Endovascular Repair) randomized trial were used to develop the risk score. Variables considered included age, sex, haemodynamic markers and aortic morphology. Backwards selection was used to identify relevant predictors. Predictive performance was assessed using calibration plots and the C-statistic. Validation of the newly developed and other previously published scores was conducted in four external populations. The net benefit of treating patients based on a risk threshold compared with treating none was quantified. Results: Data from 536 patients in the IMPROVE trial were included. The final variables retained were age, sex, haemoglobin level, serum creatinine level, systolic BP, aortic neck length and angle, and acute myocardial ischaemia. The discrimination of the score for 48-h mortality in the IMPROVE data was reasonable (C-statistic 0·710, 95 per cent c.i. 0·659 to 0·760), but varied in external populations (from 0·652 to 0·761). The new score outperformed other published risk scores in some, but not all, populations. An 8 (95 per cent c.i. 5 to 11) per cent improvement in the C-statistic was estimated compared with using age alone. Conclusion: The assessed risk scores did not have sufficient accuracy to enable potentially life-saving decisions to be made regarding intervention. Focus should therefore shift to offering repair to more patients and reducing non-intervention rates, while respecting the wishes of the patient and family