No description available.

Apolipoprotein C-I (apoC-I) plays an important role in the metabolism of plasma triglyceride levels and cholesterol metabolism. Little is known about the regulation of apoC-I production by human adipocytes. Aim. To investigate the effect of different tissue culture conditions on the synthesis and secretion of apoC-I and apoE in human SW872 liposarcoma cells and to study the effects of apoC-I overexpression in these same cells. Methods. SW872 cells were grown in DMEM/F-12 (3:1, v/v). QPCR was used to quantify mRNA synthesis. ELISAs were used to quantify intracellular and extracellular proteins. Colorimetric reaction kits were used to analyze intracellular cholesterol and triglyceride concentrations. Results . Maturation experiments revealed that after 17 days in culture, SW872 cells contained significantly more cholesterol (100%) and triglyceride (3-fold). Cell maturation was associated with significantly higher levels of apoE mRNA (+200%) but not apoC-I mRNA (-50%). The cells secreted more apoC-I (+110%) and apoE (+340%). Cellular apoC-I increased 620% and apoE increased 1540%. Treatment of cells during maturation with insulin (0, 10 or 1000 nM) significantly reduced the secretion of apoC-I and apoE (-14% and -56%, respectively) and intracellular apoC-I and apoE (-10% and -12%, respectively. Overexpression of apoC-I in SW872 cells resulted in increased cell number (+70%) and decreased lipids per cell (-32% triglyceride, -36% cholesterol) as compared to controls. Conclusion. These results suggest that apoC-I and apoE production is differentially regulated at the transcriptional level in adipocytes and that apoC-I and apoE play a role in the maturation of human adipocytes and may have an important role in mediating or regulating cell lipid accumulation. As well, overexpression of apoC-I in SW872 cells impedes cellular lipid accumulation and stimulates cellular proliferation.

Atherosclerosis, hardening and narrowing of the arteries, is the principal underlying cause of heart attacks, strokes, and peripheral vascular disease, which kills more than 600,000 Americans each year. High plasma levels of low-density lipoproteins (LDL) are linked to the formation of atherosclerotic plaques in arteries. LDL is the last metabolic product of very low-density lipoprotein (VLDL), which is secreted from the liver along with one molecule of apolipoprotein B (apoB). Current therapies to control levels of LDL include: cholesterol synthesis inhibitors or statins, low-fat diets and antisense oligonucleotides to reduce cholesterol levels. Recent studies recommend lower clinical levels of plasma LDL to maintain an individual’s health, especially of those who have already developed atheroscle- rotic plaques. However, existing therapies are often unable to achieve these aggressive limits. Furthermore, patients have shown various levels of intolerance to these treatments. In order to develop new, targeted drugs, that can control LDL levels with minimal side effects, it is imperative to understand, in detail, the process of apoB-containing lipoprotein formation. ApoB is one of the largest human proteins known (4563 residues) and previous attempts to solve the structure have been unsuccessful, mainly due to analyzing the protein as a whole or by large sections. To advance the field we will go by a different approach. I present here a construct that represents roughly 8% of the whole protein, apoB9-17 (residues 430 to 782). This section of the protein is believed to play a pivotal role in the assembly process of LDL. My hypothesis is that this construct will be well-behaved and suitable for structural and functional analysis. The study shows that apoB9-17 can be produced in considerable quantities from bacterial cells and can be purified by means of a 6-histidine tag with a good yield. Furthermore, circular dichroism analysis shows the construct contains the expected secondary structure at room temperature and is stable at a wide temperature range (50 to 70 ◦C) at low concentrations. The construct here described will be useful to test the effect of mutations such as the one found in patients with Familial hypobetalipoproteinemia (FHBL). Furthermore, this construct contains two regions believed to be of vital importance for LDL particle formation: the alpha-helical region (residues 430 to 570) is believed to associate with MTP at the initial stages of LDL formation and the c-sheet (residues 614 to 782), which may form part of the lipid recruiting process. Both essential aspects to ultimately develop therapies that can modulate VLDL particle formation.

No description available.

Sphingolipids have been implicated as potential atherogenic lipids. Inhibition of hepatic serine palmitoyl transferase (SPT) reduces plasma sphingomyelin (SM) levels leading to a reduction of atherosclerosis in apolipoprotein-E gene knockout (apoE-/-) mice. In my thesis I have investigated the possibility that the reduced atherosclerosis resulting from SPT inhibition is associated with decreases in plasma glycosphingolipids (GSL). Furthermore, I examined whether SPT inhibition can lead to regression of atherosclerotic lesions. This thesis shows the SPT inhibitor myriocin inhibits atherosclerosis in apoE-/- mice fed a high fat diet. Lesion inhibition was most pronounced at the aortic arch and distal sites of the thoracic and abdominal aorta. There was also a trend towards a reduction in lesion area at the aortic root. Myriocin treatment resulted in significant reductions in both plasma SM and GSL concentrations. Moreover, it was demonstrated that myriocin significantly inhibited the progression of established atherosclerosis. Although the inhibition of lesion progression was observed mainly in the distal regions of the aorta, regression of lesion size was not detected. In addition, this thesis demonstrated for the first time that selective inhibition of GSL synthesis by the GSL synthesis inhibitor, d-threo-1- ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4) had no significant impact on lesion area in apoE-deficient mice. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, this thesis indicates that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo. In other studies we assessed the possibility that myriocin may also be acting to increase hepatic apoA-I production via the inhibition of ERK phosphorylation. To address this, HepG2 cells and primary mouse hepatocytes were treated with myriocin. This significantly increased apoA-I mRNA, and protein levels. It also increased apoA-I secrection, and decreased ERK phosphorylation. These in vitro data indicate that ERK phosphorylation plays a role in modulating apoA-I expression, and that myriocin???s mechanism of action is linked to this pathway. Overall, this thesis has expanded the current literature regarding the role of sphingolipid synthesis inhibition and atherosclerosis.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O cÃncer à uma doenÃa grave que atinge milhares de pessoas no mundo, possuindo altos Ãndices de morbidade e mortalidade. Atualmente existem diversos alvos terapÃuticos no tratamento do cÃncer, um deles reside no fato de inibir a replicaÃÃo do DNA impossibilitando a cÃlula tumoral de se duplicar como à o caso do 5-fluoruracil (5-FU).O objetivo do trabalho foi avaliar o efeito antiinflamatÃrio do peptÃdeo mimÃtico da ApoE (COG 133) em camundongos Swiss desafiados pela mucosite intestinal induzida pelo 5-FU. Utilizamos camundongos Swiss machos com peso de 30g, sendo desafiados com injeÃÃo (dose Ãnica) intraperitoneal de 5-FU (450 mg/kg).Alguns animais foram tratados com diferentes doses (0,3, 1,0 e 3,0 ÂM) do peptÃdeo mimÃtico da apolipoproteÃna E (ApoE) COG133 por via intra-peritoneal, apÃs o desafio com 5-FU. Animais tratados com PBS foram usados como controles. Os camundongos foram sacrificados com soluÃÃo de deslocamento cervical 3 dias apÃs o desafio com 5-FU.Algumas amostras de intestino foram congeladas imediatamente em nitrogÃnio lÃquido e em seguida armazenadas em freezer a -80ÂC para biologia molecular. Outras amostras foram fixadas em formaldeÃdo para processamento histolÃgico.Monitoramos o peso corporal dos animais e fizemos leucometria a partir de coleta retrorbital de sangue para avaliaÃÃo do efeito citotÃxico mielosupressor do 5-FU nos grupos experimentais.Avaliamosos parÃmetros morfomÃtricos de altura de vilo e profundidade de cripta em segmentos do duodeno. Para a detecÃÃo de proteÃnas de interesse, utilizamos o Western blot com a utilizaÃÃo dos seguintes anticorpos IL-1&#946;, TNF-&#945; e iNOS. Para o RT-PCR, avaliamos os seguintes primers IL-1&#946;, TNF-&#945; e iNOS. No ensaio de citocinas por ELISA, utilizamos os seguintes anticorpos IL-1&#946;, IL-10, TNF-&#945;. Nas anÃlises morfomÃtricas de duodeno, encontramos uma grande reduÃÃo na altura de vilos e profundidade de criptas nosanimais tratados com 5-FU, que foi reduzida com com a utilizaÃÃo do peptÃdeo COG133 na dose 3,0 ÂM (p<0,001).Nas anÃlises histopatolÃgicas, observamos um intensoinfiltrado inflamatÃrio nos animais do grupo que foi injetadocomo 5-FU, que foi parcialmente revertido pela administraÃÃo do peptÃdeo COG133.Os dados de ELISA evidenciaram um aumento da quantidade de citocinas prÃ-inflamatÃrias IL-1&#946; (p<0,05), TNF-&#945; (p<0,05) e uma reduÃÃo da citocina antiinflamatÃria IL-10 (p<0,05), quando comparados ao controle salina, em duodenos de animais que foram injetados com 5-FU. A administraÃÃo do peptÃdeo COG 133 reduziu os nÃveis de IL-1&#946; de maneira estatisticamente significante (p<0,05 1&#956;M; p<0,001 3&#956;M), TNF-&#945; (p<0,001 na dose de 1,0 &#956;M); p<0,001 na dose de 3&#956;M) e para IL-10 (p<0,001 na dose de 3&#956;M).Dados de Western blot evidenciam que o peptÃdeo na dose de 3&#956;M reduziu de maneira estatisticamente significante a expressÃo de citocinas como IL-1&#946; (p<0,001), TNF-&#945; (p<0,05) e do mediador inflamatÃrio iNOS (p<0,05) no duodeno. Pelo protocolo de RT-PCR,encontramos uma elevaÃÃo na expressÃo do transcrito para TNF-&#945; e iNOS nos animais injetados com 5-FU, o que foi parcialmente diminuÃdo pela administraÃÃo do peptÃdeo na dose de 3&#956;M para TNF-&#945; (p<0,05) e iNOS (p<0,05)pelo ANOVA.A partir dos nossos achados, podemos concluir que o peptÃdeo mimÃtico da ApoE (COG 133) possui uma aÃÃo anti-inflamatÃria, especialmente no segmento duodeno, visto que reduz a expressÃo de citocinas prÃ-inflamatÃrias de animais desafiados, dessa forma sugerindo sua utilizaÃÃo em humanos com mucosite intestinal sob quimio/radioterapia.

No description available.

Poster exhibited at GPSC Student Showcase, November 7th, 2014, University of Arizona. / Given the early emergence of Alzheimer’s disease (AD) related pathology in Down syndrome (DS; Trisomy 21), it is possible that changes may be evident in childhood or adolescence in Apolipoprotein (APOE) e3/e4 or e4/e4 genotypes in relation to e3/e3 genotypes. Given findings of early involvement of striatum amyloid beta (Aβ) peptide deposition in DS, we propose that a profile of executive and inhibitory control dysfunction will be found in youth carrying the risk e4 allele. From a pool of 72 children and adolescents with DS we examined a sub-sample with the risk e4 allele (n = 8; e3/e4) and without the risk e4 allele (n = 8; e3/e3). Participants were matched for age and ethnicity (range 8 - 21 years; mean age 14 years). Karyotypes were gathered from medical records, confirming a diagnosis of Trisomy 21. We collected genetic information (Oragene saliva kit) in home; they were sent to the Emory Biomarker Service Center to determine genotypes. We administered the Kaufman Brief Intelligence Test (KBIT-2) and a set of cognitive outcomes measures validated for Down syndrome, the Arizona Cognitive Test Battery. Results from the KBIT-2 indicated no significant differences in verbal raw score (p = 0.65), non-verbal raw score (p = 0.69), or intelligence quotient (IQ) (p = 0.32). Neuropsychological test scores did differ; with poorer performance in the e4 sample on the CANTAB Paired Associates Learning task (p = 0.05) and parent/caregiver reports of working memory (p = 0.08). Therefore, as early as adolescence some changes may be seen in e4 carriers.

Background: Carriers of the APOE epsilon 4 allele are at increased risk of developing Alzheimer's disease (AD), and have been shown to have reduced cerebral metabolic rate of glucose (CMRgl) in the same brain areas frequently affected in AD. These individuals also exhibit reduced plasma levels of apolipoprotein E (apoE) attributed to a specific decrease in the apoE4 isoform as determined by quantification of individual apoE isoforms in APOE epsilon 4 heterozygotes. Whether low plasma apoE levels are associated with structural and functional brain measurements and cognitive performance remains to be investigated. Methods: Using quantitative mass spectrometry we quantified the plasma levels of total apoE and the individual apoE3 and apoE4 isoforms in 128 cognitively normal APOE epsilon 3/epsilon 4 individuals included in the Arizona APOE cohort. All included individuals had undergone extensive neuropsychological testing and 25 had in addition undergone FDG-PET and MRI to determine CMRgl and regional gray matter volume (GMV). Results: Our results demonstrated higher apoE4 levels in females versus males and an age-dependent increase in the apoE3 isoform levels in females only. Importantly, a higher relative ratio of apoE4 over apoE3 was associated with GMV loss in the right posterior cingulate and with reduced CMRgl bilaterally in the anterior cingulate and in the right hippocampal area. Additional exploratory analysis revealed several negative associations between total plasma apoE, individual apoE isoform levels, GMV and CMRgl predominantly in the frontal, occipital and temporal areas. Finally, our results indicated only weak associations between apoE plasma levels and cognitive performance which further appear to be affected by sex. Conclusions: Our study proposes a sex-dependent and age-dependent variation in plasma apoE isoform levels and concludes that peripheral apoE levels are associated with GMV, CMRgl and possibly cognitive performance in cognitively healthy individuals with a genetic predisposition to AD.

Thesis (Ph. D.)--University of Chicago, Dept. of Chemistry, June 1999. / Includes bibliographical references. Also available on the Internet