Reconstituted low density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes to cells

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3β-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37°C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Vascular smooth muscle mitochondria: Magnesium content and transport

Endogenous magnesium content and magnesium transport of isolated bovine vascular smooth muscle mitochondria were studied. Mitochondria isolated from atherosclerotic bovine arteries contained two to three times as much magnesium (178 nmol/mg of mitochondrial protein) as those isolated from normal arteries (67 nmol/mg of mitochondrial protein). Electron-opaque granules were visible in the unstained unfixed mitochondria and could be shown with electron probe analysis to consist of magnesium, calcium, and phosphorus. At concentrations of external Mg²⁺ from 0 to 6 mm, the vascular smooth muscle mitochondria exhibited respiratory substrate-supported release of Mg²⁺ as studied with metallochromic indicator, eriochrome blue, using dual-wavelength spectrophotometry. The maximal velocity of energized release (3 nmol of Mg²⁺/s/mg of mitochondrial protein) was observed at 4 mm external Mg²⁺ and the half-maximal transport occurred at 0.5 mm.     

Mice lacking the mitochondrial exonuclease MGME1 develop inflammatory kidney disease with glomerular dysfunction

Mitochondrial DNA (mtDNA) maintenance disorders are caused by mutations in ubiquitously expressed nuclear genes and lead to syndromes with variable disease severity and tissue-specific phenotypes. Loss of function mutations in the gene encoding the mitochondrial genome and maintenance exonuclease 1 (MGME1) result in deletions and depletion of mtDNA leading to adult-onset multisystem mitochondrial disease in humans. To better understand the in vivo function of MGME1 and the associated disease pathophysiology, we characterized a Mgme1 mouse knockout model by extensive phenotyping of ageing knockout animals. We show that loss of MGME1 leads to de novo formation of linear deleted mtDNA fragments that are constantly made and degraded. These findings contradict previous proposal that MGME1 is essential for degradation of linear mtDNA fragments and instead support a model where MGME1 has a critical role in completion of mtDNA replication. We report that Mgme1 knockout mice develop a dramatic phenotype as they age and display progressive weight loss, cataract and retinopathy. Surprisingly, aged animals also develop kidney inflammation, glomerular changes and severe chronic progressive nephropathy, consistent with nephrotic syndrome. These findings link the faulty mtDNA synthesis to severe inflammatory disease and thus show that defective mtDNA replication can trigger an immune response that causes age-associated progressive pathology in the kidney.     

The C1B domains of novel PKCε and PKCη have a higher membrane binding affinity than those of the also novel PKCδ and PKCθ

The C1 domains of novel PKCs mediate the diacylglycerol-dependent translocation of these enzymes. The four different C1B domains of novel PKCs (δ, ε, θ and η) were studied, together with different lipid mixtures containing acidic phospholipids and diacylglycerol or phorbol ester. The results show that either in the presence or in the absence of diacylglycerol, C1Bε and C1Bη exhibit a substantially higher propensity to bind to vesicles containing negatively charged phospholipids than C1Bδ and C1Bθ. The observed differences between the C1B domains of novel PKCs (in two groups of two each) were also evident in RBL-2H3 cells and it was found that, as with model membranes, in which C1Bε and C1Bη could be translocated to membranes by the addition of a soluble phosphatidic acid without diacylglycerol or phorbol ester, C1Bδ and C1Bθ were not translocated when soluble phosphatidic acid was added, and diacylglycerol was required to achieve a detectable binding to cell membranes. It is concluded that two different subfamilies of novel PKCs can be established with respect to their propensity to bind to the cell membrane and that these peculiarities in recognizing lipids may explain why these isoenzymes are specialized in responding to different triggering signals and bind to different cell membranes.     

Probiotics as an emerging therapeutic strategy to treat NAFLD: focus on molecular and biochemical mechanisms

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, both in adults and in children. NAFLD is characterized by aberrant lipid storage in hepatocytes (hepatic steatosis) and inflammatory progression to nonalcoholic steatohepatitis. Evidences so far suggest that intrahepatic lipid accumulation does not always derive from obesity. Gut microbiota has been considered as a regulator of energy homeostasis and ectopic fat deposition, suggesting its implications in metabolic diseases. Probiotics are live microbial that alter the enteric microflora and have beneficial effects on human health. Although the molecular mechanisms of probiotics have not been completely elucidated yet, many of their effects have proved to be beneficial in NAFLD, including the modulation of the intestinal microbiota, an antibacterial substance production, an improved epithelial barrier function and a reduced intestinal inflammation. Given the close anatomical and functional correlation between the bowel and the liver, and the immunoregulatory effects elicited by probiotics, the aim of this review is to summarize today's knowledge about probiotics in NAFLD, focusing in particular on their molecular and biochemical mechanisms, as well as highlighting their efficacy as an emerging therapeutic strategy to treat this condition.     

Large-scale preventive chemotherapy for the control of helminth infection in Western Pacific countries: six years later

In 2001, Urbani and Palmer published a review of the epidemiological situation of helminthiases in the countries of the Western Pacific Region of the World Health Organization indicating the control needs in the region. Six years after this inspiring article, large-scale preventive chemotherapy for the control of helminthiasis has scaled up dramatically in the region. This paper analyzes the most recent published and unpublished country information on large-scale preventive chemotherapy and summarizes the progress made since 2000. Almost 39 million treatments were provided in 2006 in the region for the control of helminthiasis: nearly 14 million for the control of lymphatic filariasis, more than 22 million for the control of soil-transmitted helminthiasis, and over 2 million for the control of schistosomiasis. In general, control of these helminthiases is progressing well in the Mekong countries and Pacific Islands. In China, despite harboring the majority of the helminth infections of the region, the control activities have not reached the level of coverage of countries with much more limited financial resources. The control of food-borne trematodes is still limited, but pilot activities have been initiated in China, Lao People's Democratic Republic, and Vietnam.     

A stapled chromogranin A-derived peptide homes in on tumors that express αvβ6 or αvβ8 integrins

Rationale: The αvβ6- and αvβ8-integrins, two cell-adhesion receptors upregulated in many tumors and involved in the activation of the latency associated peptide (LAP)/TGFβ complex, represent potential targets for tumor imaging and therapy. We investigated the tumor-homing properties of a chromogranin A-derived peptide containing an RGDL motif followed by a chemically stapled alpha-helix (called “5a”), which selectively recognizes the LAP/TGFβ complex-binding site of αvβ6 and αvβ8.Methods: Peptide 5a was labeled with IRDye 800CW (a near-infrared fluorescent dye) or with ¹⁸F-NOTA (a label for positron emission tomography (PET)); the integrin-binding properties of free peptide and conjugates were then investigated using purified αvβ6/αvβ8 integrins and various αvβ6/αvβ8 single - or double-positive cancer cells; tumor-homing, biodistribution and imaging properties of the conjugates were investigated in subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, and in mice bearing subcutaneous αvβ8-positive prostate tumors.Results: In vitro studies showed that 5a can bind both integrins with high affinity and inhibits cell-mediated TGFβ activation. The 5a-IRDye and 5a-NOTA conjugates could bind purified αvβ6/αvβ8 integrins with no loss of affinity compared to free peptide, and selectively recognized various αvβ6/αvβ8 single- or double-positive cancer cells, including cells from pancreatic carcinoma, melanoma, oral mucosa, bladder and prostate cancer. In vivo static and dynamic optical near-infrared and PET/CT imaging and biodistribution studies, performed in mice with subcutaneous and orthotopic αvβ6-positive carcinomas of the pancreas, showed high target-specific uptake of fluorescence- and radio-labeled peptide by tumors and low non-specific uptake in other organs and tissues, except for excretory organs. Significant target-specific uptake of fluorescence-labeled peptide was also observed in mice bearing αvβ8-positive prostate tumors.Conclusions: The results indicate that 5a can home to αvβ6- and/or αvβ8-positive tumors, suggesting that this peptide can be exploited as a ligand for delivering imaging or anticancer agents to αvβ6/αvβ8 single- or double-positive tumors, or as a tumor-homing inhibitor of these TGFβ activators.