Detection of information flow between cortical laminae activities and auditory evoked potentials in rats by means of Granger causality.

We seek to understand the relation between invasive high-resolution data and non-invasive measurement in an animal model in an auditory sensory adaptation experimental setting. In a previous study, we estimated the mutual information between the phase of auditory evoked responses (AER) with the phase of local field potentials (LFP) of auditory cortices at different frequency ranges. The results showed a consistently high level of information sharing between the AER activities as well as the responses from the granular layer, which was known as the main thalamo-recipient layer. However, mutual information was fundamentally an undirected measure of information flow. In this study we investigated how well we could characterize direction of information flow, by using Granger causality (GC), between different cortical laminae and functional projections on to the AER activities. We obtained that based on the GC coefficients, we are able to extract the connectivity between different cortical laminae to some extend and also a strong connection between the AER and granular layer. In our future study, we would like to construct a reliable picture of network connectivity, both functionally and anatomically, between different layers at more specified frequencies and much finer temporal resolutions.

Oncogenic Ras differentially regulates metabolism and anoikis in extracellular matrix-detached cells.

In order for cancer cells to survive during metastasis, they must overcome anoikis, a caspase-dependent cell death process triggered by extracellular matrix (ECM) detachment, and rectify detachment-induced metabolic defects that compromise cell survival. However, the precise signals used by cancer cells to facilitate their survival during metastasis remain poorly understood. We have discovered that oncogenic Ras facilitates the survival of ECM-detached cancer cells by using distinct effector pathways to regulate metabolism and block anoikis. Surprisingly, we find that while Ras-mediated phosphatidylinositol (3)-kinase signaling is critical for rectifying ECM-detachment-induced metabolic deficiencies, the critical downstream effector is serum and glucocorticoid-regulated kinase-1 (SGK-1) rather than Akt. Our data also indicate that oncogenic Ras blocks anoikis by diminishing expression of the phosphatase PHLPP1 (PH Domain and Leucine-Rich Repeat Protein Phosphatase 1), which promotes anoikis through the activation of p38 MAPK. Thus, our study represents a novel paradigm whereby oncogene-initiated signal transduction can promote the survival of ECM-detached cells through divergent downstream effectors.

Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration.

Inflammatory breast cancer (IBC) is a rare and highly invasive type of breast cancer, and patients diagnosed with IBC often face a very poor prognosis. IBC is characterized by the lack of primary tumor formation and the rapid accumulation of cancerous epithelial cells in the dermal lymphatic vessels. Given that normal epithelial cells require attachment to the extracellular matrix (ECM) for survival, a comprehensive examination of the molecular mechanisms underlying IBC cell survival in the lymphatic vessels is of paramount importance to our understanding of IBC pathogenesis. Here we demonstrate that, in contrast to normal mammary epithelial cells, IBC cells evade ECM-detachment-induced apoptosis (anoikis). ErbB2 and EGFR knockdown in KPL-4 and SUM149 cells, respectively, causes decreased colony growth in soft agar and increased caspase activation following ECM detachment. ERK/MAPK signaling was found to operate downstream of ErbB2 and EGFR to protect cells from anoikis by facilitating the formation of a protein complex containing Bim-EL, LC8, and Beclin-1. This complex forms as a result of Bim-EL phosphorylation on serine 59, and thus Bim-EL cannot localize to the mitochondria and cause anoikis. These results reveal a novel mechanism that could be targeted with innovative therapeutics to induce anoikis in IBC cells.

Multimodal optical, X-ray CT, and SPECT imaging of a mouse model of breast cancer lung metastasis.

Tumor heterogeneity is recognized as a major issue within clinical oncology, and the concept of personalized molecular medicine is emerging as a means to mitigate this problem. Given the vast number of cancer types and subtypes, robust pre-clinical models of cancer must be studied to interrogate the molecular mechanisms involved in each scenario. In particular, mouse models of tumor metastasis are of critical importance for pre-clinical cancer research at the cancer cell molecular level. In many of these experimental systems, tumor cells are injected intravenously, and the distribution and proliferation of these cells are subsequently analyzed via ex vivo methods. These techniques require large numbers of animals coupled with time-consuming histological preparation and analysis. Herein, we demonstrate the use of two facile and noninvasive imaging techniques to enhance the study of a pre-clinical model of breast cancer metastasis in the lung. Breast cancer cells were labeled with a near-infrared fluorophore that enables their visualization. Upon injection into a living mouse, the distribution of the cells in the body was detected and measured using whole animal fluorescence imaging. X-ray computed tomography (CT) was subsequently used to provide a quantitative measure of longitudinal tumor cell accumulation in the lungs over six weeks. A nuclear probe for lung perfusion, 99mTc-MAA, was also imaged and tested during the time course using single photon emission computed tomography (SPECT). Our results demonstrate that optical fluorescence methods are useful to visualize cancer cell distribution patterns that occur immediately after injection. Longitudinal imaging with X-ray CT provides a convenient and quantitative avenue to measure tumor growth within the lung space over several weeks. Results with nuclear imaging did not show a correlation between lung perfusion (SPECT) and segmented lung volume (CT). Nevertheless, the combination of animal models and noninvasive optical and CT imaging methods provides better research tools to study cancer cell differences at the molecular level. Ultimately, the knowledge gleaned from these improved studies will aid researchers in uncovering the mechanisms mediating breast cancer metastasis, and eventually improve the treatments of patients in the clinic.

Basal and post-methionine serum homocysteine and lipoprotein abnormalities in patients with chronic liver disease.

BACKGROUND: Lipoprotein abnormalities are commonly found in chronic liver diseases (CLDs), particularly hypercholesterolemia in primary biliary cirrhosis (PBC). However, affected patients may not be at increased risk of coronary heart disease. Cirrhotic patients display impaired methionine clearance, and an increased level of homocysteine, a methionine metabolite, is an independent risk factor for coronary heart disease. Thus, we hypothesized that the low risk of coronary heart disease in patients with CLD may be related to low serum levels of homocysteine. The aim of this study was to test this hypothesis after methionine load and to describe the serum lipoprotein profile in patients with PBC and in patients with hepatocellular liver disease. METHODS: Fifteen female patients (mean age, 58.2 +/- 11.7 years) with PBC, 15 female patients (mean age, 54.5 +/- 9.6 years) with other causes of CLD, and 15 healthy sex- and age-matched controls were given L-methionine (50 mg/kg of ideal body weight). Basal fasting serum homocysteine level and 2, 4, and 6 hours of post-methionine load were determined using high-performance liquid chromatography with a fluorometric detector. Levels of fasting serum cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), lipoprotein (a) (Lp(a)), and apoprotein B were also determined. RESULTS: Results showed that mean basal and post-methionine load (6 hours) serum homocysteine levels were statistically significantly higher in the patients with PBC and with CLD than in the control group (P=0.04) and that levels of serum cholesterol, LDL, HDL, and apoprotein B were significantly higher in the PBC patients than in the other two groups (P < or = 0.05). There was no correlation between any of these parameters and the severity of liver disease. Serum HDL was significantly lower in the CLD group (P < or = 0.05) and correlated with severity of liver disease. There was no significant difference in serum cholesterol, LDL, or apoprotein B between the CLD group and the controls. Serum triglyceride and Lp(a) levels were similar for all three groups. CONCLUSIONS: In contrast to previous reports, the site of the methionine metabolic impairment was found to be below the homocysteine synthesis level. For most patients with CLD, factors other than serum homocysteine or Lp(a) are responsible for the reduction in the risk of coronary heart disease. Further studies with larger samples are needed.

Effect of prolonged fasting on plasma lipids, lipoproteins and apolipoprotein B in 12 physicians participating in a hunger strike: an observational study.

BACKGROUND: Dyslipidemia and obesity serve as risk factors for the development of atherosclerotic cardiovascular disease. Fasting is sometimes recommended for treating these conditions. This study was undertaken to try to resolve conflicting results reported in the literature. OBJECTIVES: To study the effect of fasting (0 calories, with free intake of fluids) for 3-5 days on plasma concentration of triglyceride, cholesterol and apolipoprotein B. METHODS: Physicians, about to begin a hunger strike, were divided into four groups: normolipidemic non-obese men (group 1), two moderately obese men and two men with type IV hyperlipidemia (group 2), healthy non-obese women (group 3), and healthy non-obese women on oral contraceptives (group 4). Adherence to fasting was monitored daily by detailed interviews, loss of weight, drop in plasma glucose, presence of ketonuria, progressive rise in serum creatinine and uric acid, and decrease in plasma pH. We monitored their serum glucose, electrolytes, liver function, lipids, lipoproteins and apolipoprotein B on days 0, 3, and 5. RESULTS: Physicians who adhered to complete fasting lost more than 1.5% of their body weight after 3 days of fasting (n = 12), and more than 3.2% at 5 days (n = 5). All non-obese normolipidemic males and females (groups 1 and 3) showed an increase in plasma triglyceride (by 28-162%) and very low density lipoprotein cholesterol (by 22-316%) after 3 days of fasting. The obese and hyperlipidemic men (group 2) showed a decrease of 17-63% in their VLDL cholesterol, and the women on oral contraceptives (group 4) showed a 20% decrease in their plasma triglyceride on day 3. Low density lipoprotein cholesterol increased by 13% in group 2, decreased by 7.3% in group 4, and remained unchanged in group 1 and 3. Apolipoprotein B level correlated well with LDL cholesterol in all groups. High density lipoprotein cholesterol changes were inconsistent. CONCLUSIONS: These results help to explain and reconcile previous published reports. The metabolic background of the individual together with the amount of energy consumed affect the behavior of plasma lipids and lipoproteins levels during fasting.

Diurnal heterogeneity in structure and function of low density lipoproteins of normolipidemic males.

Structural changes in low density lipoproteins (LDL) have been shown to alter their metabolism and atherogenic potential. We investigated the diurnal changes in size and composition of LDL in seven healthy, non-obese, normolipidemic male volunteers consuming a standard diet (14.5% protein, 31.9% fat, 53.6% carbohydrate and 383 mg cholesterol/day) and continuing their daily routine. The food was divided into three meals and three snacks, and blood samples were obtained at 7 AM (after 12 h fasting), noon, 8 PM, midnight and 3 AM. LDL were isolated by both sequential and density gradient ultracentrifugation (d = 1.019 - 1.050 g/ml), and analyzed for lipids, apolipoproteins, size, and affinity to LDL receptors. Diurnal LDL preparations differ from fasting LDL in both chemical and physical parameters. The former get richer in triglyceride (TG/cholesterol weight ratio 0.23 vs. 0.16), larger in diameter (21.2 +/- 0.2 vs. 22.4 +/- 0.1 nm), and enriched in a more buoyant fraction (74.0 +/- 4.6 vs. 41.9 +/- 3.8% of LDL cholesterol in d = 1.019 - 1.035 g/ml). These structural changes in LDL were associated with enhanced affinity to LDL receptors in both human skin fibroblasts and HepG2 cells, as demonstrated by competition experiments with fasting human 125I-LDL. The observed diurnal heterogeneity in both the structure and the function of LDL may be attributed to the absorptive state as it did not occur during prolonged fasting. These diurnal changes may be important for better understanding LDL metabolism in vivo and for the elucidation of the atherogenic process.

Copper-induced lipid oxidation in unfractionated plasma: the lag preceding oxidation as a measure of oxidation-resistance.

In spite of the pathological significance of oxidation of plasma lipids, no method is currently available for the evaluation of the susceptibility of these lipids to oxidation in unfractionated plasma. Here we demonstrate that copper-induced oxidation of diluted plasma, in the presence of citrate can be monitored continuously by recording the absorbance at 245 nm. The kinetics of accumulation of oxidation products in unfractionated plasma is a sum of lipid oxidation products obtained in low and high density lipoproteins isolated from the same plasma. The kinetic profiles are reproducible and can be performed with plasma samples even after prolonged storage at 4 degrees C (up to two months) or after freezing and thawing of the plasma. Being simple and reproducible, yet correlating with the oxidizability of low and high density lipoproteins, this method can be used to evaluate the "oxidation resistance" of plasma lipids and thus serve as a standard index of the susceptibility of the plasma lipids of patients to oxidation-inflicted pathologies, including atherosclerosis.

Diurnal changes in plasma lipoproteins of free-living healthy men consuming a standard diet.

Although it is well established that plasma lipoproteins are involved in atherogenesis, most patients with coronary heart disease have normal or nearly normal fasting plasma lipid and lipoprotein levels. As most of the human life is spent in the fed state we elected to investigate the changes occurring in plasma lipids and lipoproteins during normal daily activities. We studied 12 healthy, normolipidemic and non-obese subjects on a standard diet divided into three meals and three snacks. This method was found to be accurate and reproducible in four subjects who underwent repeated studies. The major diurnal changes were observed in plasma triglyceride level and in the composition of plasma lipoproteins. The degree and pattern of these changes differed among subjects but was specific and constant for each individual. The extent of these diurnal changes, which correlated positively with fasting plasma triglyceride and inversely with plasma HDL2 levels, may play an important role in the pathogenesis of atherosclerosis.

Heterogeneity in peritoneal transport during continuous ambulatory peritoneal dialysis and its impact on ultrafiltration, loss of macromolecules and plasma level of proteins, lipids and lipoproteins.

We investigated the effect of heterogeneity in the peritoneal transport of plasma proteins on dialysis efficiency and plasma levels of proteins, lipids and lipoproteins in 32 patients undergoing long-term continuous ambulatory peritoneal dialysis (CAPD; 9 females and 23 males, 18-76 years old). Eleven patients were studied on several occasions (at 0-42 months) and the remainder at 6-60 months on CAPD (n = 49). We have divided our patients arbitrarily into two equal groups according to their protein concentration in the peritoneal effluent at the end of an 8-hour cycle. Patients with a high peritoneal protein concentration (> or = 2 g/l/1.73 m2) have lower ultrafiltration capacity, higher glucose absorption rate and increased loss of most plasma proteins [including albumin, immunoglobulins (Ig), complement components and high-density lipoproteins (HDL)] compared to patients with a low peritoneal protein concentration (< 2 g/l/1.73 m2). Consequently, plasma levels of albumin, IgM and HDL were lower in patients with a high peritoneal protein concentration. The latter had also increased levels of plasma triglycerides and very-low-density lipoproteins. The difference observed in peritoneal transport between the groups could be ascribed only in part to the duration of CAPD treatment, and hence to the number of peritonitis episodes but not to medications. Therefore, we suggest that inherent constitutional factors may be responsible for some of the observed heterogeneity in the peritoneal transport of these patients which is already evident at the start of CAPD treatment. Patients with high peritoneal transport are exposed to an augmented atherogenic plasma lipid profile in addition to a reduction in dialysis efficiency (ultrafiltration failure). These patients may become prone also to nutritional and immunological disturbances. Therefore, we suggest taking these effects into consideration before choosing the appropriate dialysis modality in patients with increased peritoneal transport for plasma proteins.

Regulation of lipoprotein lipase secretion in murine macrophages during foam cell formation in vitro. Effect of triglyceride-rich lipoproteins.

Triglyceride rich-lipoproteins induce triglyceride accumulation in macrophages, leading to foam cell formation. The correlation between cell triglyceride accumulation and lipoprotein lipase (LPL) secretion in murine macrophages and the role that LPL plays in the accumulation process were examined. LPL secretion is defined as the extracellular LPL activity that accumulates during a 4-hour incubation of treated and untreated cells in a bovine serum albumin-containing RPMI-1640 medium. LPL secretion was suppressed (up to 70%) in a dose- and time-dependent manner when J774.1 cells were incubated with chylomicrons, very low density lipoproteins, and intermediate density lipoproteins but not with low or high density lipoproteins from normolipidemic and hypertriglyceridemic subjects. Oleic acid both suppressed LPL secretion and invoked triglyceride accumulation. Suppression of LPL secretion preceded gross triglyceride accumulation, was reversible, and was not the result of a reduction in LPL mRNA. P388D1 cells neither secreted LPL nor accumulated triglyceride. Inhibition of LPL secretion by tunicamycin in both peritoneal macrophages and J774.1 cells prevented a hypertriglyceridemic very low density lipoprotein-induced triglyceride accumulation, an effect that was counteracted by addition of exogenous LPL. The results suggest that 1) extracellular hydrolysis of lipoprotein triglyceride is a major factor in inducing foam cell formation and 2) LPL secretion may be regulated by cell energy needs, and when these needs are exceeded, LPL secretion is suppressed.

Kinetics of peritoneal protein loss during CAPD: I. Different characteristics for low and high molecular weight proteins.

We studied the peritoneal protein loss in 13 patients during CAPD using 2 liters of 1.5% dextrose dialysis solutions. We compared the kinetic characteristics of the peritoneal mass transfer and clearance of proteins over a wide range of molecular size, to those of small molecular weight solutes. The peritoneal clearance of all studied proteins and solutes correlated strongly and negatively with their molecular mass. No changes were observed in these clearances during 58 months of dialysis. Unlike the peritoneal mass transfer and clearance of small molecular weight solutes (less than 200) which revealed a remarkable progressive drop after the first hour of an eight-hour dialysis cycle, the mass transfer and clearance of proteins of large molecular weight (greater than 68,000) was continuous throughout the eight hours. The clearance of proteins of small molecular weight (less than 15,000) showed similar kinetics to small solutes (less than 200). These results indicate that long dwell times (6 or 8 hr) of peritoneal dialysis are detrimental for the loss of large molecular weight proteins (such as albumin and immunoglobulins) in view of the negligible dialysance of both small solutes (creatinine and potassium) and "intermediate molecules" (represented by the small molecular weight proteins) during the latter hours of long dwell cycles. Thus we suggest that substituting CAPD (3 x 8 hr or 4 x 6 hr) with CCPD (6 x 1 hr) may limit protein loss in these patients.

Kinetics of peritoneal protein loss during CAPD: II. Lipoprotein leakage and its impact on plasma lipid levels.

We quantified the plasma levels and peritoneal loss of lipids and lipoproteins, and studied the composition of plasma and effluent lipoproteins in 16 patients on CAPD (5 females and 11 males, 18 to 76 years old). Five patients were studied prospectively (at 0, 1, 3 and 6 months) and 11 patients at 6 to 58 months on CAPD (N = 30). Elevated levels of plasma VLDL and reduced levels of plasma HDL were maintained in these patients throughout 58 months of CAPD, whereas the initially increased LDL levels showed a tendency towards normalization. All plasma lipoproteins (VLDL, IDL, LDL and HDL) were present in the peritoneal effluent. The lipoproteins isolated from plasma and peritoneal fluid shared a similar lipid and apolipoprotein composition. The peritoneal transport characteristics of plasma lipoproteins were similar to other plasma macromolecules. Their clearance correlated with their molecular mass, plasma concentration and dwell time, but was not affected by duration of CAPD treatment. The plasma lipid and lipoprotein levels were unaffected by the rate of glucose absorption. The peritoneal protein clearance correlated positively with plasma levels of triglyceride and LDL, and negatively with plasma HDL. An inverse correlation was observed also between plasma levels of HDL and its peritoneal clearance (r = -0.393, P less than 0.025, N = 30). The continuous peritoneal loss of HDL and the hypertriglyceridemia were found to contribute most to the persistent low plasma levels of HDL in CAPD patients, and thus may lead to the accelerated atherosclerosis observed in these patients.

Interactions between human and carp (Cyprimus carpio) low density lipoproteins (LDL) and LDL receptors.

1. We have compared the concentration and chemical composition of carp and human plasma lipoproteins and studied their interaction with human fibroblast LDL receptors. 2. The main lipoproteins in carp are of high density (HDL) in contrast to low density lipoproteins (LDL) in human. 3. Carp lipoproteins are devoid of apolipoprotein (apo) E, a major ligand for interaction with LDL receptors in mammals. 4. Carp very low density lipoproteins (VLDL) and LDL but not HDL nor apoA-I cross react with human LDL in their interaction with LDL receptors on human cultured fibroblasts. 5. Carp liver membranes possess high affinity receptors that are saturable and have calcium dependent ligand specificity (apoB and apoE) similar to human LDL receptor. Carp VLDL and LDL but not HDL nor its major apolipoprotein complexed to L-alpha-phosphatidylcholine dimyristoyl (apoA-I-DMPC) competed with the specific binding of human LDL to this receptor.