Physiology

MARIANA NIKOLOVA KARAKASHIAN, Ph.D. Associate Professor Ph.D. Bulgarian Academy of Science, 1992 Office: MS-579 Medical Center 0298 Tel: (859) 323-8210 Lab: MS-531 Tel: (859) 323-1115 E-mail: mnikolo@uky.edu Curriculum Vita (pdf)

Our Laboratory studies the function and regulation of metabolism of sphingolipids, a class of second messengers that participate in cellular response to stress and inflammation. We use multifaceted approach that includes the following specific projects:

Project 1: Role of ceramide in up-regulation of acute phase proteins during inflammation and aging.

Acute phase proteins like C-reactive protein, a1 acid glycoprotein and serum Amyloid A are markers and independent indicators for inflammation, aging and aging-associated disease like atherosclerosis. Ceramide, which is generated during numerous stress conditions including aging and acute phase response of liver to inflammation is part of the signaling cascade leading to induction of mRNA levels of these proteins. The major focus of this project is to understand the mechanisms that regulate ceramide generation during inflammation and aging and how the excess ceramide affects the cellular responses.

Our in vitro approach includes the use of (i) adenovirus-mediate gene transfer to overexpress candidate sphingomyelinases in primary hepatocytes and hepatocellular cell lines, (ii) different cell biology approaches to evaluate their subcellular localization, and (iii) state-of-the art analytical techniques to quantify the mass of sphingolipids. The end-point analyses in this project are different methods to assess the level of mRNA including RT-PCR, the activity of transcription factors from the C/EBP, NFkB and Ap-1 families as well as the activation state of other down-stream components of the signaling cascade, including MAP kinases.

In parallel, we apply an in vivo approach involving studies on the acute phase response in acid sphingomyelinase knockout mice.

Project 2: Role of ceramide in circulating lipoproteins for endothelial dysfunction and apoptosis.

In addition to being intracellular second messengers, ceramide is present in serum lipoproteins and its levels are up-regulated during systemic inflammation and other stress conditions. The goal of this project is to identify the functional significance of these increases. The experimental approach that we use includes (i) experiments on isolation and characterization of serum lipoproteins from humans and rodents, (ii) induction of specific modification of the levels of ceramide in Low-density lipoproteins using biophysical tools, (iii) investigation on up-take and metabolism of ceramide enriched LDL by human microvascular cells, and (iv) the cellular consequences of ceramide-enriched lipoproteins on cell survival and apoptosis. Knockout mice are also used to confirm the conlusions from cell culture studies in physiological context.

Techniques often used in the lab:

Biochemical: Western blot, RT-PCR, gel-shift analyses, protein purification and enzyme activity assays, immunoprecipitation, lipid extractions, TLC, HPLC.

Cell culture: Maintaining of different cell lines, isolation of primary hepatocytes from rats and mice.

Molecular biology: Adenovirus-mediated gene transfer in mammalian cells, cloning of different ceramide metabolizing enzymes in various plasmids, infection of mammalian cells, and genotyping of mice.

Cell biology: Fluorescent microscopy of subcellular organelles, indirect immunofluorescence, TUNEL, Annexin V, Hoechst staining, Flow cytometry. Sub-cellular fractionation. Receptor internalization and ligand-receptor interactions.

Work with small animals: Breeding, animal genotyping and identification.

Recent publications:

Rutkute K, Asmis RH, Nikolova-Karakashian MN. Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation. J Lipid Res. 48(11):2443-52, 2007.

Rutkute K, Karakashian AA, Giltiay NV, Dobierzewska A, Nikolova-Karakashian MN. Aging in rat causes hepatic hyperresposiveness to interleukin-1beta which is mediated by neutral sphingomyelinase-2. Hepatology. 46(4):1166-1176, 2007.

Rutkute K, Nikolova-Karakashian MN. Regulation of insulin-like growth factor binding protein-1 expression during aging. Biochem Biophys Res Commun. 361(2): 263-9, 2007.

Karakashian, A.A., Giltiay, N.V., Smith G.M., and Nikolova-Karakashian, M.N. Expression of Neutral Sphingomyelinase-2 (NSMase-2) in primary rat hepatocytes: possible role in regulation of IL-1ß induced activation of JNK. FASEB J, April 1, 2004 (e-pub ahead of print).

Boyanovsky, B., Karakashian, A., King, K. Giltyay, N., and Nikolova-Karakashian, M. Ceramide-enriched low-density lipoproteins induce apoptosis in human microvascular endothelial cells. J. Biol. Chem. 2003, (In press).

Other Publications