Comparative effects of trichothecene mycotoxins on bovine platelet function: Acetyl T-2 toxin,a more potent inhibitor than T-2 toxin

The effects of the trichothecene mycotoxins (acetyl T-2 toxin, T-2 toxin, HT-2 toxin, palmityl T-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), and T-2 tetraol) on bovine platelet function were examined in homologous plasma stimulated with platelet activating factor (PAF). The mycotoxins inhibited platelet function with the following order of potency: acetyl T-2 toxin > palmityl T-2 toxin = DAS > HT-2 toxin = T-2 toxin. While T-2 tetraol was completely ineffective as an inhibitor, DON exhibited minimal inhibitory activity at concentrations above 10×10^?4M. The stability of the platelet aggregates formed was significantly reduced in all mycotoxin treated platelets compared to that of the untreated PAF controls. It is suggested that the increased sensitivity of PAF stimulated bovine platelets to the more lipophilic mycotoxins may be related to their more efficient partitioning into the platelet membrane compared to the more hydrophilic compounds.     

Proteasome alterations during adipose differentiation and aging: links to impaired adipocyte differentiation and development of oxidative stress

Intracellular proteins are degraded by a number of proteases, including the ubiquitin-proteasome pathway (UPP). Impairments in the UPP occur during the aging of a variety of tissues, although little is known in regards to age-related alterations to the UPP during the aging of adipose tissue. The UPP is known to be involved in regulating the differentiation of a variety of cell types, although the potential changes in the UPP during adipose differentiation have not been fully elucidated. How the UPP is altered in aging adipose tissue and adipocyte differentiation and the effects of proteasome inhibition on adipocyte homeostasis and differentiation are critical issues to elucidate experimentally. Adipogenesis continues throughout the life of adipose tissue, with continual differentiation of preadipocytes essential to maintaining tissue function during aging, and UPP alterations in mature adipocytes are likely to directly modulate adipose function during aging. In this study we demonstrate that aging induces alterations in the activity and expression of principal components of the UPP. Additionally, we show that multiple changes in the UPP occur during the differentiation of 3T3-L1 cells into adipocytes. In vitro data link observed UPP alterations to increased levels of oxidative stress and altered adipose biology relevant to both aging and differentiation. Taken together, these data demonstrate that changes in the UPP occur in response to adipose aging and adipogenesis and strongly suggest that proteasome inhibition is sufficient to decrease adipose differentiation, as well as increasing oxidative stress in mature adipocytes, both of which probably promote deleterious effects on adipose aging.     

Cloning, expression, and purification of the Staphylococcus simulans lysostaphin using the intein-chitin-binding domain (CBD) system

The Staphylococcus simulans gene encoding lysostaphin has been PCR amplified from pRG5 recombinant plasmid (ATCC 67076) and cloned into Escherichia coli expression pTYB12 vector (IMPACT-CN System, New England BioLabs) which allows the overexpression of a target protein as a fusion to a self-cleavable affinity tag. The self-cleavage activity of the intein allows the release of the lysostaphin enzyme from the chitin-bound intein tag, resulting in a single-column purification of the target protein. This abundant overproduction allows purifying milligram amounts of the enzyme.     

High Dietary Fat Selectively Increases Catalase Expression within Cardiac Mitochondria

Obesity is a predictor of diabetes and cardiovascular disease. One consequence of obesity is dyslipidemia characterized by high blood triglycerides. It has been proposed that oxidative stress, driven by utilization of lipids for energy, contributes to these diseases. The effects of oxidative stress are mitigated by an endogenous antioxidant enzyme network, but little is known about its response to high fat utilization. Our experiments used a multiplexed quantitative proteomics method to measure antioxidant enzyme expression in heart tissue in a mouse model of diet-induced obesity. This experiment showed a rapid and specific up-regulation of catalase protein, with subsequent assays showing increases in activity and mRNA. Catalase, traditionally considered a peroxisomal protein, was found to be present in cardiac mitochondria and significantly increased in content and activity during high fat feeding. These data, coupled with the fact that fatty acid oxidation enhances mitochondrial H₂O₂ production, suggest that a localized catalase increase is needed to consume excessive mitochondrial H₂O₂ produced by increased fat metabolism. To determine whether the catalase-specific response is a common feature of physiological conditions that increase blood triglycerides and fatty acid oxidation, we measured changes in antioxidant expression in fasted versus fed mice. Indeed, a similar specific catalase increase was observed in mice fasted for 24 h. Our findings suggest a fundamental metabolic process in which catalase expression is regulated to prevent damage while preserving an H₂O₂-mediated sensing of diet composition that appropriately adjusts insulin sensitivity in the short term as needed to prioritize lipid metabolism for complete utilization.     

吐鲁番市胃食管反流病发生相关危险因素分析（附280例报道）

目的：探讨280例胃食管反流病（GERD）的分布特点及危险因素。方法：对临床诊断和胃镜确诊的280例GERD患者进行临床和风险因子相关性分析。结果：不论汉族还是维族,男性患者比例均明显高于女性;汉族患者高发年龄段早于维族患者（z=-2．939,P=0．003,）;汉族和维族患者占反流性食管炎和Barrett食管比例分别为42．4％、81_3％及56．5％、18．8％,其中汉族患者Barrett食管比例较高（X2=14．358,P=0．000）;肥胖、习惯性便秘、重体力活动者、饮食习惯不良在维族患者中的比例较高（P〈0．001）。结论：GERD与性别、年龄密切相关,男性多于女性,汉族患者发病年龄高峰旱于维族患者;汉族患者Barrett食管发生比例高于维族患者;肥胖、习惯性便秘、重体力活动、饮食习惯不良可能是GERD尤其是维族人群GERD的危险因素。     

Vulnerability of synaptosomes from apoE knock-out mice to structural and oxidative modifications induced by A beta(1-40): implications for Alzheimer's disease

Apolipoprotein E (apoE) plays an important role in the response to central nervous system injury. The e4 allele of apoE and amyloid beta-peptide (Abeta) are associated with Alzheimer's disease (AD) and may be central to the pathogenesis of this disorder. Recent studies demonstrate evidence for neurodegeneration and increased lipid peroxidation in transgenic mice lacking apoE (KO). In the current study, synaptosomes were prepared from apoE KO mice to determine the role of apoE in synaptic membrane structure and to determine susceptibility to oxidative damage by Abeta(1-40). ApoE KO mice exhibited structural modifications to lipid and protein components of synaptosomal membranes as determined by electron paramagnetic resonance in conjunction with lipid- and protein- specific spin labels. Incubation with 5 microM Abeta(1-40) resulted in more severe oxidative modifications to proteins and lipids in apoE KO synaptosomes as measured by protein carbonyls, an index of protein oxidation, and TBARs and protein-bound 4-hydroxynonenal (HNE), markers of lipid oxidation. Together, these data support a role for apoE in the modulation of oxidative injury and in the maintenance of synaptic integrity and are discussed with reference to alterations in AD brain.     

Selective protection by stably transfected human ALDH3A1 (but not human ALDH1A1) against toxicity of aliphatic aldehydes in V79 cells

Toxic medium chain length alkanals, alkenals, and 4-hydroxyalkenals that are generated during lipid peroxidation are potential substrates for aldehyde dehydrogenase (ALDH) isoforms. We have developed transgenic cell lines to examine the potential for either human ALDH1A1 or ALDH3A1 to protect against damage mediated by these toxic aldehydes. Using crude cytosols from stably transfected cell lines, these aldehydes were confirmed to be excellent substrates for ALDH3A1, but were poorly oxidized by ALDH1A1. Expression of ALDH3A1 by stable transfection in V79 cells conferred a high level of protection against growth inhibition by the medium-chain length aldehyde substrates with highest substrate activity, including hexanal, trans-2-hexenal, trans-2-octenal, trans-2-nonenal, and 4-hydroxy-2-nonenal (HNE). This was reflected in a parallel ability of ALDH3A1 to prevent depletion of glutathione by these aldehydes. Expression of hALDH3 completely blocked the potent induction of apoptosis by HNE in both V79 cells and in a RAW 264.7 murine macrophage cell line, consistent with the observed total prevention of HNE-protein adduct formation. Structure-activity studies indicated that the rank order of potency for the contributions of HNE functional groups to toxicity was aldehyde >/=C2=C3 double bond>C4-hydroxyl group. Oxidation of the aldehyde moiety of HNE to a carboxyl by ALDH3A1 expressed in stably transfected cell lines drastically reduced its potency for growth inhibition and apoptosis induction. In contrast, ALDH1A1 expression provided only moderate protection against trans-2-nonenal (t2NE), and none against the other six-nine carbon aldehydes. Neither ALDH1A1 nor ALDH3A1 conferred any protection against acrolein, acetaldehyde, or chloroacetaldehyde. A small degree of protection against malondialdehyde was afforded by ALDH1A1, but not ALDH3A1. Paradoxically, cells expressing ALDH3A1 were 1.5-fold more sensitive to benzaldehyde toxicity than control V79 cells. These studies demonstrate that expression of class 3 ALDH, but not class 1 ALDH, can be an important determinant of cellular resistance to toxicity mediated by aldehydes of intermediate chain length that are produced during lipid peroxidation.     

Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme

Pyrococcus woesei (DSM 3773) alpha-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)alpha-amyl and pYTB2alpha-amyl vectors obtained were used for expression of thermostable alpha-amylase or fusion of alpha-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of alpha-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation-they exhibit only 35% of total cell activity-and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable alpha-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75 degrees C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95 degrees C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90 degrees C and 110 degrees C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120 degrees C. Maltose was the main end product of starch hydrolysis catalyzed by this alpha-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.