C ellular oxidative stress is one of the factors responsible for the propagation of liver diseases, such as hepatitis, cirrhosis, and hepatoma. 1 Several primary antioxidant defense systems such as superoxide dismutase (SOD), catalase, glutathione (GSH), and glutathione peroxidase are present intracellularly. These systems scavenge reactive oxygen species (ROS) such as hydrogen peroxide, superoxide, lipid peroxides, and free radicals. Exposure to oxidative stress may, however, deplete the cellular antioxidant capacity. Therefore, other antioxidant defense systems are expected to play an important role in oxidative stress.Liver fatty acid binding protein (L-FABP) is a 14-kd protein found abundantly in the cytoplasm and the nucleus of hepatocytes. 2,3 L-FABP is very likely to be an effective endogenous antioxidant, because it has high affinity and capacity to bind long-chain fatty acid oxidation products. 4,5 Hepatocyte L-FABP concentration could be as high as 0.4 mmol/L, 6 and it contains a large number of reducing amino acid residues (1 cysteine and 7 methionine residues) in its molecular structure. With an accessible volume enclosed by the molecular surface of L-FABP of 28,600 Å 3,7 the concentration of total methionine residues in L-FABP could be as high as approximately 400 mmol/L. Methionine and cysteine amino acids are regarded as cellular scavengers of activated xenobiotics and

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Accurate diagnosis of mosquito allergy has been precluded by the difficulty of obtaining salivary allergens. In this study, we expressed, purified, characterized and investigated the clinical relevance of a recombinant Aedes aegypti salivary allergen, rAed a 1. Two cDNA segments were ligated together to form the full-length Aed a 1 gene. rAed a 1 was expressed using a baculovirus/insect cell system, and purified using a combination of anion-exchange and gel-filtration chromatography. The purified rAed a 1 bound to human IgE, as detected by ELISA, ELISA inhibition tests and immunoblot analyses. Epicutaneous tests with rAed a 1 and a commercial whole-body AE: aegypti extract, and AE: aegypti bite tests were performed in 48 subjects. Nine of 31 (29%) of the subjects with positive immediate bite tests also had a positive rAed a 1 immediate skin reaction and 32% had an positive immediate test to the commercial extract. Six of 33 (18%) of the subjects with positive delayed bite tests also had a positive rAed a 1 delayed skin reaction and 6% had a positive delayed test to the commercial extract. Furthermore, rAed a 1-induced flare sizes significantly correlated with mosquito bite-induced flare sizes. None of the subjects with negative bite tests had a positive skin test to rAed a 1 or to commercial extract. We conclude that the rAed a 1 has identical antigenicity and biological activity to native Aed a 1, can be used in the in vitro and in vivo diagnosis of mosquito allergy, and is more sensitive than mosquito whole-body extract for detecting delayed skin reactions.

Abstract-Hyperglycemia and dyslipidemia are two biochemical markers of diabetes mellitus. Increased incidence of cardiovascular disease and impaired fibrinolytic activity have been found in diabetic subjects. Previous studies have demonstrated that low density lipoproteins (LDLs) stimulate the production of plasminogen activator inhibitor-1 (PAI-1) and reduce the generation of tissue plasminogen activator (tPA) in vascular endothelial cells (ECs). The present study investigated the effect of glycated LDL on the production of PAI-1 and tPA in cultured human umbilical vein ECs (HUVECs). Glycation increased the abundance of glucitollysine and conjugated dienes in LDL and amplified the overproduction of PAI-1 and the reduction in tPA generation from HUVECs induced by LDL. The steady-state levels of PAI-1 mRNA in glycated LDL-treated ECs were significantly higher than those in native LDL-treated cells. Actinomycin D blocked the increase in PAI-1 generation induced by glycated LDL. Glycated LDL did not significantly reduce the levels of tPA mRNA but attenuated de novo synthesis of tPA in ECs. Treatment with 25 mmol/L aminoguanidine, an antioxidant and inhibitor of the formation of advanced glycation end products, during glycation normalized glycated LDL-induced generation of PAI-1 and tPA in ECs. The results of the present study indicate that glycation enhances the production of PAI-1 and attenuates tPA synthesis in ECs induced by LDL, which may contribute to the increased incidence of cardiovascular complications in diabetes. Formation of advanced glycation end products or peroxidation may be involved in glycated LDL-induced alterations in the generation of fibrinolytic regulators from ECs.

Excessive platelet activation and accumulation can lead to vessel occlusion and thus present major therapeutic challenges in cardiovascular medicine. Apyrase, an ecto-enzyme with ADPase and ATPase activities, rapidly metabolizes ADP and ATP released from platelets and endothelial cells, thereby reducing platelet activation and recruitment. In the present study, we expressed a 68-kDa recombinant mosquito (Aedes aegypti) salivary apyrase using a baculovirus/insect cell expression system and purified it to homogeneity using anion-exchange chromatography on a large scale. A yield of 18 mg of purified recombinant apyrase was obtained from 1 litre of the medium. Kinetic analysis indicated that the recombinant apyrase had a K(m) of 12.5 microM for ADP and a K(m) of 15.0 microM for ATP. The recombinant apyrase inhibited ADP-, collagen- and thrombin-induced human platelet aggregation in a dose-dependent manner, indicating that the recombinant protein retained nucleotidase activity in a whole cell system, which suggests that it may serve as a therapeutic agent for inhibition of platelet-mediated thrombosis.

To determine whether hepatocyte membrane potential differences (PDs) are depolarized in human HCC and whether depolarization is associated with changes in GABA A receptor expression, hepatocyte PDs and ␥-aminobutyric acid (GABA) A receptor messenger RNA (mRNA) and protein expression were documented in HCC tissues via microelectrode impalement, real-time reverse-transcriptase polymerase chain reaction, and Western blot analysis, respectively. HCC tissues were significantly depolarized (؊19.8 ؎ 1.3 versus ؊25. In recent in vitro studies, we demonstrated that restoration of depolarized malignant hepatocyte cell membrane potential differences (PDs) toward those documented in nonmalignant hepatocytes results in a loss of malignant features, including decreased proliferative activity, absence of colony formation in soft agar, and normalization of phenotypic appearance. 2 Whether HCC tissues are depolarized relative to adjacent nontumor tissues and the mechanisms whereby such depolarization might exist have yet to be reported.

Increasing hepatocyte membrane potentials by augmenting GABAergic activity inhibits nonmalignant hepatocyte proliferative activity. The objectives of this study were to document 1) potential differences (PDs) of four malignant hepatocyte cell lines, 2) GABAA receptor mRNA expression in the same cell lines, and 3) effects of restoring malignant hepatocyte PDs to levels approximating those of resting, nonmalignant hepatocytes. Hepatocyte PDs were documented in nonmalignant and malignant (Chang, HepG2, HuH-7, and PLC/PRF/5) hepatocytes with a fluorescent voltage-sensitive dye and GABAA receptor expression by RT-PCR and Western blot analyses. Compared with nonmalignant human hepatocytes, all four malignant cell lines were significantly depolarized (P < 0.0001, respectively). Only PLC/PRF/5 cells had detectable GABAA-beta3 receptor mRNA expression and all cell lines were negative for GABAA-beta3 receptor protein by Western blot analysis. Stable transfection of Chang cells with GABAA-beta3 receptor cDNA resulted in significant increases in PD and decreases in proliferative activity as manifest by decreased [3H]thymidine and bromodeoxyurieine incorporation rates, 4-[3-(4-lodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate activity, a lower mitotic index, prolongation of cell-doubling times, and attenuated growth patterns compared with cells transfected with vector alone. Colony formation in soft agar and the number of abnormal mitoses were also significantly decreased in GABAA-beta3 receptor transfected cells. The results of this study indicate 1) relative to healthy hepatocytes, malignant hepatocytes are significantly depolarized, 2) GABAA-beta3 receptor expression is absent in malignant hepatocyte cell lines, and 3) increasing the PD of malignant hepatocytes is associated with less proliferative activity and a loss of malignant features.

Allergic reactions to mosquito bites are an increasing clinical concern. Due to lack of availability of mosquito salivary allergens, they are under-diagnosed. Here, we reported a newly cloned mosquito Aedes (Ae.) aegypti salivary allergen and its clinical relevance. A cDNA encoding a 30-kDa Ae. aegypti salivary protein, designated Aed a 3, was isolated from an expression library. The full-length cDNA was cloned into baculovirus expression vector and recombinant Aed a 3 (rAed a 3) was expressed, purified and characterized. Skin prick tests with purified rAed a 3, and Ae. aegypti mosquito bite tests were performed in 43 volunteers, some of whom had serum rAed a 3-specific IgE levels measured. The primary nucleotide sequence, deduced amino acid sequence, and IgE binding sites of Aed a 3 were identified. rAed a 3-selected antibodies recognized a 30 kDa Ae. aegypti saliva protein. rAed a 3 bound IgE in mosquito-allergic volunteers and the binding could be inhibited by addition of natural mosquito extract dose-dependently. Immediate skin test reactions to rAed a 3 correlated significantly with reactions produced by mosquito bites. Of the bite-test positive volunteers, 32% had a positive rAed a 3 skin test and 42% had specific IgE. No bite-test negative volunteers reacted to rAed a 3 in either the skin tests or the IgE assays, confirming the specificity of the assay. Aed a 3 is a major mosquito salivary allergen. Its recombinant form has biological activity and is suitable for use in skin tests and serum IgE assays in mosquito-allergic individuals.