Expression of bovine adrenodoxin in E. coli and site-directed mutagenesis of /2 Fe-2S/ cluster ligands.

Expression systems for adrenodoxin into the periplasm and the cytoplasm of E. coli have been developed as a prerequisite for site-directed mutagenesis studies. In both systems the /2Fe-2S/ cluster of the protein was correctly assembled, the cytoplasmic one gives, however, a tenfold higher expression level. To determine which of the five cysteines at positions 46, 52, 55, 92, and 95 coordinate the /2Fe-2S/ center, they have been individually mutated into serines. From these mutants, only C95S forms a functionally active holoprotein. Thus, residues 46, 52, 55, and 92 are the cysteines that coordinate the /2Fe-2S/ cluster in adrenodoxin.     

Disappearance and reformation of the nuclear lamina structure during specific stages of meiosis in oocytes

The nuclear lamina is a rigid, proteinaceous layer underlying the inner nuclear membrane of eucaryotic cells. It is present in somatic cell nuclei, disappears during mitosis, and is absent from male meiotic cells. We have investigated the disappearance and reformation of the nuclear lamina during meiosis in oocytes, using immunofluorescence and electron microscopy. We find that the status of the nuclear lamina during meiosis of oocytes differs from the reversible depolymerization seen in mitosis in two respects. First, the lamina disappears during meiotic prophase without affecting the structure of the nuclear membranes or the nuclear pores. Second, the proteins of the dissociated lamina are undetectable by immunological methods in pachytene oocytes, whereas they persist in the cytoplasm during mitosis.     

Top-Down Lipidomics Reveals Ether Lipid Deficiency in Blood Plasma of Hypertensive Patients

Background

Dyslipoproteinemia, obesity and insulin resistance are integrative constituents of the metabolic syndrome and are major risk factors for hypertension. The objective of this study was to determine whether hypertension specifically affects the plasma lipidome independently and differently from the effects induced by obesity and insulin resistance.

Methodology/Principal Findings

We screened the plasma lipidome of 19 men with hypertension and 51 normotensive male controls by top-down shotgun profiling on a LTQ Orbitrap hybrid mass spectrometer. The analysis encompassed 95 lipid species of 10 major lipid classes. Obesity resulted in generally higher lipid load in blood plasma, while the content of tri- and diacylglycerols increased dramatically. Insulin resistance, defined by HOMA-IR >3.5 and controlled for BMI, had little effect on the plasma lipidome. Importantly, we observed that in blood plasma of hypertensive individuals the overall content of ether lipids decreased. Ether phosphatidylcholines and ether phosphatidylethanolamines, that comprise arachidonic (20∶4) and docosapentaenoic (22∶5) fatty acid moieties, were specifically diminished. The content of free cholesterol also decreased, although conventional clinical lipid homeostasis indices remained unaffected.

Conclusions/Significance

Top-down shotgun lipidomics demonstrated that hypertension is accompanied by specific reduction of the content of ether lipids and free cholesterol that occurred independently of lipidomic alterations induced by obesity and insulin resistance. These results may form the basis for novel preventive and dietary strategies alleviating the severity of hypertension.     

Behavioral treatment of irritable bowel syndrome: A 1-year follow-up study

Sixteen clients afflicted with irritable bowel syndrome (IBS) were reassessed 1 year following completion of a multicomponent treatment package incorporating progressive muscle relaxation, thermal biofeedback, cognitive therapy, and IBS education. For the 14 patients who kept a 2-week symptom diary, significant reductions in ratings of abdominal pain and tenderness, diarrhea, and flatulence were obtained comparing pretreatment and follow-up symptom-diary ratings. Eleven of 14 clients were improved over pretreatment levels, 57% met the criteria for clinical improvement of at least a 50% reduction in major symptom scores, and all but 1 of 16 rated themselves as subjectively improved.     

PCSK9 inhibition fails to alter hepatic LDLR,circulating cholesterol,and atherosclerosis in the absence of ApoE

LDL cholesterol (LDL-C) contributes to coronary heart disease. Proprotein convertase subtilisin/kexin type 9 (PCSK9) increases LDL-C by inhibiting LDL-C clearance. The therapeutic potential for PCSK9 inhibitors is highlighted by the fact that PCSK9 loss-of-function carriers exhibit 15–30% lower circulating LDL-C and a disproportionately lower risk (47–88%) of experiencing a cardiovascular event. Here, we utilized pcsk9^−/− mice and an anti-PCSK9 antibody to study the role of the LDL receptor (LDLR) and ApoE in PCSK9-mediated regulation of plasma cholesterol and atherosclerotic lesion development. We found that circulating cholesterol and atherosclerotic lesions were minimally modified in pcsk9^−/− mice on either an LDLR- or ApoE-deficient background. Acute administration of an anti-PCSK9 antibody did not reduce circulating cholesterol in an ApoE-deficient background, but did reduce circulating cholesterol (−45%) and TGs (−36%) in APOE*3Leiden.cholesteryl ester transfer protein (CETP) mice, which contain mouse ApoE, human mutant APOE3*Leiden, and a functional LDLR. Chronic anti-PCSK9 antibody treatment in APOE*3Leiden.CETP mice resulted in a significant reduction in atherosclerotic lesion area (−91%) and reduced lesion complexity. Taken together, these results indicate that both LDLR and ApoE are required for PCSK9 inhibitor-mediated reductions in atherosclerosis, as both are needed to increase hepatic LDLR expression.     

Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols.

A variety of eukaryotic cell surface proteins, including the variant surface glycoproteins of African trypanosomes, rely on a covalently attached lipid, glycosylphosphatidylinositol (GPI), for membrane attachment. GPI anchors are synthesized in the endoplasmic reticulum by stepwise glycosylation of phosphatidylinositol (via UDP-GlcNAc and dolichol-P-mannose) followed by the addition of phosphoethanolamine. The experiments described in this paper are aimed at identifying the biosynthetic origin of the terminal phosphoethanolamine group. We show that trypanosome GPIs can be labelled via CDP-[3H]ethanolamine or [beta-32P]CDP-ethanolamine in a cell-free system, indicating that phosphoethanolamine is acquired en bloc. In pulse-chase experiments with CDP-[3H]ethanolamine we show that the GPI phosphoethanolamine is not derived directly from CDP-ethanolamine, but instead from a relatively stable metabolite, such as phosphatidylethanolamine (PE), generated from CDP-ethanolamine in the cell-free system. To test the possibility that PE is the immediate donor of the GPI phosphoethanolamine moiety, we describe metabolic labelling experiments with [3H]serine and show that GPIs can be labelled in the absence of detectable radiolabelled CDP-ethanolamine, presumably via [3H]PE generated from [3H]phosphatidylserine (PS). The data support the proposal that the terminal phosphoethanolamine group in trypanosome GPIs is derived from PE.