Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

Resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-infected T-cell lines

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-kappaB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-kappaB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-lLnorleucinal (LLnL), an NF-kappaB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-kappaB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-kappaB may be a useful adjunct in clinical use of Apo2L against ATL.     

A Novel Assay System for Anti-Human Immunodeficiency Virus Type 1 (HIV-1) Activity Using a Subclone of a Monocytic Cell Line,U937

A subclonal cl.1–14 cell was established from a monocytic cell line U937 by a limiting dilution method. The anti-HIV-1 activity of some antiviral compounds was evaluated in HIV-1-infected cl.1–14 cells. The results demonstrated that although AZT was a potent inhibitor of HIV-1 replication in cl.1–14 cells, its 50% effective concentration (EC₅₀) values was 80 times higher than that in HIV-1 infected MT-4 cells; the EC₅₀ of AZT was 0.16 μM and 0.002 μM in cl.1–14 and MT-4 cells, respectively. In contrast, the anti-HIV-1 activity of ddA, ddI and ddC in cl.1–14 cells was comparable to that in MT-4 cells. The antiviral activity of nevirapine, dextran sulfate, curdlan sulfate and T22 did not differ significantly between the cl. 1–14 and MT-4 cells. The antiviral activity of several compounds in the HIV-1-infected cl.1–14 cells was similar to that in the HIV-1jr -fl -infected human peripheral macrophages. Our results suggest that cl.1–14 cell cultures are very useful for estimating antiviral activity and more advantageous than the use of peripheral blood macrophages.     

Mechanistic studies of an antibody with chorismate mutase activity

Antibody 1F7 catalyzes the rearrangement of chorismate to prephenate. Its kinetic parameters are unaffected by changes in pH and display no solvent isotope effect or effects from addition of various cationic salts. These results are consistent with high-resolution structural information of 1F7 bound to a transition state analog.     

Microbial desulfurization of dibenzothiophene in the presence of hydrocarbon

The bacterium, Rhodococcus erythropolis H-2, which can utilize dibenzothiophene (DBT) as a sole source of sulfur in the presence of hydrocarbon, was isolated from soil samples. When this strain was cultivated in a medium containing 0.27 mM DBT and 40% n-tetradecane, DBT was metabolized stoichiometrically to 2-hydroxybiphenyl within 1 day. This strain grew in the presence of n-octane and longer-carbonchain hydrocarbons, but not with n-hexane, styrene, p-xylene, cyclooctane or toluene. DBT degradation proceeded in the resting cell system with lyophilized cells of this strain. The addition of n-tetradecane enhanced the reaction rate, the optimal concentration being 40%. DBT degradation occurred in the reaction mixture even in the presence of 70% n-tetradecane, whereas at concentrations above 80% n-tetradecane suppressed the degradation.     

Specific enzymatic assay method for homocysteine and its application to the screening of neonatal homocystinuria

A simple and specific enzymatic assay method for homocysteine is described. The method is based on the formation of S-adenosylhomocysteine from adenosine and homocysteine through the catalysis of S-adenosylhomocysteine hydrolase (EC 3.3.1.1), followed by its separation by high-performance liquid chromatography. The results for human blood, serum, and urine and those extracted from filter paper showed good correlation with those obtained on measurement with a conventional amino acid analyzer, which demonstrates that the method is useful for neonatal screening for homocystinuria.     

Subplate neurons foster inhibition

Previous work demonstrates an essential role of subplate neurons during ocular dominance (OD) column formation in the developing visual cortex. While inhibitory circuitry has also been shown to play an essential role in OD plasticity, the relationship between subplate neurons and the development of inhibitory circuits has been unclear. In this issue of Neuron, Kanold and Shatz provide evidence that maturation of inhibitory circuitry requires subplate neurons in the developing cortex.     

Purification, characterization and crystallization of enzymes for dibenzothiophene desulfurization

DszC and DszA, DBT monooxygenase and DBT sulfone monooxygenase, respectively, involved in dibenzothiophene (DBT) desulfurization, were purified to homogeneity from Rhodococcus erythropolis D-1. The two enzymes were crystallized and enzymologically characterized. We found a high activity of flavin reductase in the non-DBT-desulfurizing bacterium, Paenibacillus polymyxa A-1, which is essential for DszC and A activities, and purified to homogeneity and characterized the enzyme.