The LEC rat: a useful model for studying liver carcinogenesis related to oxidative stress and inflammation

Growing evidence indicates oxidative stress as a mechanism of several diseases including cancer. Oxidative stress can be defined as the imbalance between cellular oxidant species production and antioxidant capability shifted towards the former. Lipid peroxidation is one of the processes that takes place during oxidative stress. Lipid peroxidation products, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), are closely related to carcinogenesis as they are potent mutagens and they have been suggested as modulators of signal pathways related to proliferation and apoptosis, two processes implicated in cancer development. Mechanisms by which oxidative stress leads to tumor formation are still under investigation. The need of suitable in vivo models that could reflect that inflammation-related human carcinogenesis is evident. In this regard, the mutant strain Long Evans Cinnamon-like (LEC) rat provides a promising model for investigation of the relationship between hepatitis induced by oxidative stress and hepatocarcinogenesis because it has been demonstrated to develop spontaneous liver tumor formation related to copper accumulation and oxidative stress. In this review, the findings regarding oxidative stress and its relation with liver pathologies in LEC rats are discussed; we focus on the mechanisms proposed for HNE carcinogenesis.     

Inhibitory effect of oxygen accumulation on the growth of Spirulina platensis

Summary The growth of Spirulina platensis was studied in a light-limited culture under various dissolved oxygen (DO) concentrations. At high DO concentration, e.g. at 1.25 mM DO, the growth rate was decreased up to 36 % compared with that of 0.063 mM DO. The retarded growth rate at high DO concentrations seemed to be coupled with the degeneration of photosynthetic activity in terms of O₂ evolution. Under higher DO concentrations, superoxide dismutase and ascorbate peroxidase activities tended to increase, while the contents of photosynthetic pigment, such phycocyanin, carotenoid and chlorophyll-a decreased distinctly.     

Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL antagonists

5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.     

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.     

Reconstitution of the Frank-Starling mechanism in engineered heart tissues

According to the Frank-Starling mechanism, as the heart is stretched, it increases its contraction force. Reconstitution of the Frank-Starling mechanism is an important milestone for producing functional heart tissue constructs. Spontaneously contracting engineered heart tissues (EHTs) were reconstituted by growing dissociated chicken embryo cardiomyocytes in collagen matrices. Twitch and baseline tensions were recorded at precisely controlled levels of tissue strain. The EHTs showed a steep increase in twitch tension from 0.47 +/- 0.02 to 0.91 +/- 0.02 mN/mm2 as they were stretched at a constant rate (2.67% per min) from 86% to 100% of the length at which maximum twitch force was exerted. In response to a sudden stretch (3.3%), the twitch tension increased gradually (approximately 60 s) in a Gd3+-sensitive manner, suggesting the presence of stretch-activated Ca2+ channels. A large difference in baseline tension between lengthening (loading) and shortening (unloading) was also recorded. Disruption of nonsarcomeric actin filaments by cytochalasin D and latrunculin B decreased this difference. A simple mechanical model interprets these results in terms of mechanical connections between myocytes and nonmuscle cells. The experimental results strongly suggest that regulation of twitch tension in EHTs is similar to that of natural myocardium.     

HIV-1 escape from the CCR5 antagonist maraviroc associated with an altered and less-efficient mechanism of gp120-CCR5 engagement that attenuates macrophage tropism

Maraviroc (MVC) inhibits the entry of human immunodeficiency virus type 1 (HIV-1) by binding to and modifying the conformation of the CCR5 extracellular loops (ECLs). Resistance to MVC results from alterations in the HIV-1 gp120 envelope glycoproteins (Env) enabling recognition of the drug-bound conformation of CCR5. To better understand the mechanisms underlying MVC resistance, we characterized the virus-cell interactions of gp120 from in vitro-generated MVC-resistant HIV-1 (MVC-Res Env), comparing them with those of gp120 from the sensitive parental virus (MVC-Sens Env). In the absence of the drug, MVC-Res Env maintains a highly efficient interaction with CCR5, similar to that of MVC-Sens Env, and displays a relatively modest increase in dependence on the CCR5 N terminus. However, in the presence of the drug, MVC-Res Env interacts much less efficiently with CCR5 and becomes critically dependent on the CCR5 N terminus and on positively charged elements of the drug-modified CCR5 ECL1 and ECL2 regions (His88 and His181, respectively). Structural analysis suggests that the Val323 resistance mutation in the gp120 V3 loop alters the secondary structure of the V3 loop and the buried surface area of the V3 loop-CCR5 N terminus interface. This altered mechanism of gp120-CCR5 engagement dramatically attenuates the entry of HIV-1 into monocyte-derived macrophages (MDM), cell-cell fusion activity in MDM, and viral replication capacity in MDM. In addition to confirming that HIV-1 escapes MVC by becoming heavily dependent on the CCR5 N terminus, our results reveal novel interactions with the drug-modified ECLs that are critical for the utilization of CCR5 by MVC-Res Env and provide additional insights into virus-cell interactions that modulate macrophage tropism.     

Spectroscopic and kinetic properties of the oxidized intermediates of lignin peroxidase from Phanerochaete chrysosporium

Stopped-flow rapid scan techniques were used to obtain a spectrum of nearly homogeneous lignin peroxidase compound I (LiPI) under pseudo-first order conditions at the unusually low pH optimum (3.0) for the enzyme. The LiPI spectrum had a Soret band at 407 nm with approximately 60% reduced intensity and a visible maximum at 650 nm. Under steady-state conditions a Soret spectrum for lignin peroxidase compound II (LiPII) was also obtained. The Soret maximum of LiPII at 420 nm was only approximately 15% reduced in intensity compared to native LiP. Transient state kinetic results confirmed the pH independence of LiPI formation over the pH range 3.06-7.39. The rate constant was (6.5 +/- 0.2) x 10(5) M-1 S-1. Addition of excess veratryl alcohol to LiPI resulted in its reduction to LiPII with subsequent reduction of LiPII to the native enzyme. Reactions of LiPI and LiPII with veratryl alcohol exhibited marked pH dependencies. For the LiPI reaction the rate constants ranged from 2.5 x 10(6) M-1 S-1 at pH 3.06 to 4.1 x 10(3) M-1 S-1 at pH 7.39; for the LiPII reaction, 1.6 x 10(5) M-1 S-1 (pH 3.06) to 2.3 x 10(3) M-1 S-1 (pH 5.16). These single turnover experiments demonstrate directly that the pH dependence of these reactions dictates the overall pH dependence of this novel enzyme. These results are consistent with the one-electron oxidation of veratryl alcohol to an aryl cation radical by LiPI and by LiPII.