Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

From type 2 diabetes to antioxidant activity: a systematic review of the safety and efficacy of common and cassia cinnamon bark

Common (Cinnamomum verum, C. zeylanicum) and cassia (C. aromaticum) cinnamon have a long history of use as spices and flavouring agents. A number of pharmacological and clinical effects have been observed with their use. The objective of this study was to systematically review the scientific literature for preclinical and clinical evidence of safety, efficacy, and pharmacological activity of common and cassia cinnamon. Using the principles of evidence-based practice, we searched 9 electronic databases and compiled data according to the grade of evidence found. One pharmacological study on antioxidant activity and 7 clinical studies on various medical conditions were reported in the scientific literature including type 2 diabetes (3), Helicobacter pylori infection (1), activation of olfactory cortex of the brain (1), oral candidiasis in HIV (1), and chronic salmonellosis (1). Two of 3 randomized clinical trials on type 2 diabetes provided strong scientific evidence that cassia cinnamon demonstrates a therapeutic effect in reducing fasting blood glucose by 10.3%-29%; the third clinical trial did not observe this effect. Cassia cinnamon, however, did not have an effect at lowering glycosylated hemoglobin (HbA1c). One randomized clinical trial reported that cassia cinnamon lowered total cholesterol, low-density lipoprotein cholesterol, and triglycerides; the other 2 trials, however, did not observe this effect. There was good scientific evidence that a species of cinnamon was not effective at eradicating H. pylori infection. Common cinnamon showed weak to very weak evidence of efficacy in treating oral candidiasis in HIV patients and chronic salmonellosis.     

Expression of Rapeseed Microsomal Lysophosphatidic Acid Acyltransferase Isozymes Enhances Seed Oil Content in Arabidopsis

In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.With increasing environmental challenges and concerns, there is renewed interest in deriving plant-based sustainable alternatives for petroleum products, including carburants, lubricants, and industrial feed stocks. Modifying oilseed crops to produce oils of uniform composition containing fatty acids varying in chain length or possessing reactive functional groups is a primary objective (Jaworski and Cahoon, 2003), as is that of increasing the yield of seed oil (Lardizabal et al., 2008; Zheng et al., 2008). Early success in modifying seed oils to produce the more common fatty acids has been tempered by limited success in the production of high levels of unusual fatty acids (UFAs) in cultivated oilseeds (Thelen and Ohlrogge, 2002; Drexler et al., 2003). Such studies have led to the conclusion that in order to achieve levels of UFAs similar to those present in the oil of native species, enzymatic activities additional to fatty acid modification are necessary to optimize the synthesis (Mekhedov et al., 2001), stability (Eccleston and Ohlrogge, 1998), and channeling (Bafor et al., 1990) of the desired fatty acid into triacylglycerol (TAG).The synthesis of glycerolipids occurs in the cytoplasm using de novo-synthesized fatty acids exported from the plastid as acyl-CoA thioesters. The fatty acyl groups are incorporated into membrane and storage lipids by the sequential esterification of glycerol-3-phosphate by the action of glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) at sn-1 to form lysophosphatidic acid followed by lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) at sn-2 to form phosphatidic acid (PA; Somerville et al., 2000). Dephosphorylation of PA results in the formation of diacylglycerol (DAG), which in developing seeds may be directed into the production of TAG by acyl-CoA-independent reactions or by diacylglycerol acyltransferase (DAGAT; EC 2.3.1.20; Roscoe, 2005). The substrate preferences for acyl-thioesters and the selectivities for the acceptor molecules displayed by the microsomal acyltransferases play a crucial role in establishing the acyl composition of lipids (Frentzen, 1998). The TAG synthesized in most oilseeds of agronomic importance contains fatty acids that are the same as those present in cytoplasmic membrane lipids. In contrast, the seeds of species that synthesize TAGs with exotic fatty acid compositions possess microsomal acyltransferases that facilitate the incorporation of UFAs into storage lipids because of their broad GPAT and/or their selective DAGAT specificities (Wiberg et al., 1994; Frentzen, 1998). Furthermore, oilseeds characterized by TAGs that contain UFAs at sn-2 possess additional seed-specific microsomal LPAATs (Brown et al., 1995; Hanke et al., 1995; Knutzon et al., 1995) that exhibit a wide variation in substrate preference and that serve to ensure the channeling of UFAs to this position, thereby segregating incompatible fatty acids away from membrane lipids.Cloning of cDNAs from cultivated and exotic plants and the availability of entirely sequenced genomes from plant and algal species have revealed that a minimum of two classes of genes encoding microsomal LPAATs exist (Frentzen, 1998) within a larger, LPAAT-like gene family containing acyltransferases as yet functionally uncharacterized but distinct from GPATs (Roscoe, 2005). The class A microsomal LPAATs defined by Frentzen (1998) possess substrate preferences for C18:1-CoA typical of enzymes involved in membrane lipid synthesis and are ubiquitously expressed in the plant. In contrast, individual members of the class B LPAATs display preferences for distinct, unusual saturated or unsaturated acyl groups and are normally expressed in storage organs. Although class B LPAATs have been exploited to alter the stereochemical composition of rapeseed (Brassica napus) oil to permit the incorporation of modified fatty acids at sn-2 (Lassner et al., 1995; Knutzon et al., 1999), a significant increase in the total amount of UFAs was not accomplished by the expression of the class B LPAATs alone. In contrast, the transformation of rapeseed and Arabidopsis (Arabidopsis thaliana) with a yeast gene encoding a variant LPAAT, SLC1-1, capable of accepting very long chain fatty acyl (VLCFA)-CoA substrates resulted in an increase in the total VLCFAs and, unexpectedly, in total oil content (Zou et al. 1997).In our efforts to modify the fatty acid composition of oil in rapeseed, in particular to increase the content of VLCFAs, we have addressed the question of optimizing the environment for the correct functioning of LPAATs encoded by transgenes. The above studies using the various LPAAT transgenes indicate that channeling of UFAs into sn-2 of oilseed species remains problematic. The ability to obtain oils with uniform composition strongly depends on the occupancy of sn-2 by UFAs, yet the level of occupancy of sn-2 by fatty acids corresponding to the selectivity of the introduced LPAAT is variable and relatively modest. Occupancy of sn-2 is determined in part by the ability of the LPAAT encoded by the transgene to compete with the endogenous enzyme, a function of the acyl-CoA substrates available to the enzymes and the relative efficiencies of the enzymes to compete for the donor and acceptor substrates. We argued that there is latitude for the reduction of competing activities using an antisense strategy, and although microsomal LPAATs have been cloned from rapeseed, there are no reports of the characterization of the enzyme. Our objectives in this work were to identify and evaluate the potential contribution of LPAAT isozymes to TAG biosynthesis in rapeseed, thereby discerning targets for optimizing efforts to modify oils for industrial purposes. In this study, we catalogue a previously undescribed complexity in microsomal LPAAT diversity and identify a LPAAT isozyme likely to play an important role in TAG synthesis in rapeseed. In contrast to diverged LPAATs of plant origin, we demonstrate a positive effect of the overexpression of microsomal LPAATs on oil content and seed weight.     

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance.     

Trichinosis: Thiabendazole in the Treatment of 11 Cases

We have studied 11 cases of acute human trichinosis; five of these patients were treated with thiabendazole. We have reviewed the laboratory data for these patients and described their course. This study has failed to show any definite effect of thiabendazole on the course of human trichinosis at the doses used. However, thiabendazole may have decreased the infectivity of the larvae which were embedded in the muscles of our patients.     

In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b⁺ F4/80⁺ MHC-II⁺ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS⁺ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis.