镉诱导植物的自由基过氧化损伤

紫羊茅(Festuca rubra L.)有对重金属敏感(“S59”)和忍耐(“Merlin”)两个品种。以0～2.5mmol/LCd~(2 )的营养液分别培养“S59”和“Merlin”幼苗,随着Cd~(2 )浓度增加和处理时间延长,幼苗生长量相应下降。0.25mml/L Cd~(2 )对“S59”的生长抑制与0.5mmol/L Cd~(2 )对“Merlin”的抑制相似,表明“Merlin”比“S59”更能耐受Cd~(2 )的毒害。在Cd~(2 )抑制生长的同时,幼苗的MDA(丙二醛,膜脂过氧化产物)和POD(过氧化物酶)活性急剧上升,CAT(过氧化氢酶)和SOD活性降低,蛋白质含量明显下降。MDA大幅度上升是Cd~(2 )培养幼苗突出的生理变化之一,2.5mmol/L Cd~(2 )培养幼苗6d,“Merlin”的MDA为对照的209％,而“S59”为对照的243％。MDA的积累与培养幼苗的Cd~(2 )浓度成密切正相关,与幼苗的生长量呈负相关。当SOD活性平稳或略为上升时,MDA稳定;SOD活性开始下降,MDA便急剧上升。很可能自由基和MDA在Cd~(2 )毒害植物中充当了重要的角色。     

Oncogenic comparison of human papillomavirus type 58 E7 variants

Human papillomavirus 58 (HPV58) ranks the second or third in East Asian cervical cancers. Current studies on HPV58 are scarce and focus on the prototype. Previously, we identified the three most common circulating HPV58 E7 strains contained amino acid alterations: G41R/G63D (51%), T20I/G63S (22%) and T74A/D76E (14%) respectively. Among them, the T20I/G63S variant (V1) had a stronger epidemiological association with cervical cancer. We therefore suggested that V1 possessed stronger oncogenicity than the other two variants. Here, we performed phenotypic assays to characterize and compare their oncogenicities with HPV58 E7 prototype. Our results showed that overexpression of V1 conferred a higher colony‐forming ability to primary murine epithelial cells than prototype (P < 0.05) and other variants, implicating its higher immortalising potential. Further experiments showed that both V1 and prototype enhanced the anchorage‐independent growth of NIH/3T3 cells (P < 0.001), implicating their stronger transforming power than the two other variants. Moreover, they possessed an increased ability to degrade pRb (P < 0.001), which is a major effector pathway of E7‐driven oncogenesis. Our work represents the first study to compare the oncogenicities of HPV58 E7 prototype and variants. These findings deepened our understanding of HPV58 and might inform clinical screening and follow‐up strategy.     

Regulation of Vascular Tone,Angiogenesis and Cellular Bioenergetics by the 3-Mercaptopyruvate Sulfurtransferase/H2S Pathway: Functional Impairment by Hyperglycemia and Restoration by dl-α-Lipoic Acid

Hydrogen sulfide (H₂S), as a reducing agent and an antioxidant molecule, exerts protective effects against hyperglycemic stress in the vascular endothelium. The mitochondrial enzyme 3-mercaptopyruvate sulfurtransferase (3-MST) is an important biological source of H₂S. We have recently demonstrated that 3-MST activity is inhibited by oxidative stress in vitro and speculated that this may have an adverse effect on cellular homeostasis. In the current study, given the importance of H₂S as a vasorelaxant, angiogenesis stimulator and cellular bioenergetic mediator, we first determined whether the 3-MST/H₂S system plays a physiological regulatory role in endothelial cells. Next, we tested whether a dysfunction of this pathway develops during the development of hyperglycemia and μmol/L to diabetes-associated vascular complications. Intraperitoneal (IP) 3-MP (1 mg/kg) raised plasma H₂S levels in rats. 3-MP (10 1 mmol/L) promoted angiogenesis in vitro in bEnd3 microvascular endothelial cells and in vivo in a Matrigel assay in mice (0.3–1 mg/kg). In vitro studies with bEnd3 cell homogenates demonstrated that the 3-MP-induced increases in H₂S production depended on enzymatic activity, although at higher concentrations (1–3 mmol/L) there was also evidence for an additional nonenzymatic H₂S production by 3-MP. In vivo, 3-MP facilitated wound healing in rats, induced the relaxation of dermal microvessels and increased mitochondrial bioenergetic function. In vitro hyperglycemia or in vivo streptozotocin diabetes impaired angiogenesis, attenuated mitochondrial function and delayed wound healing; all of these responses were associated with an impairment of the proangiogenic and bioenergetic effects of 3-MP. The antioxidants dl-α-lipoic acid (LA) in vivo, or dihydrolipoic acid (DHLA) in vitro restored the ability of 3-MP to stimulate angiogenesis, cellular bioenergetics and wound healing in hyperglycemia and diabetes. We conclude that diabetes leads to an impairment of the 3-MST/H₂S pathway, and speculate that this may contribute to the pathogenesis of hyperglycemic endothelial cell dysfunction. We also suggest that therapy with H₂S donors, or treatment with the combination of 3-MP and lipoic acid may be beneficial in improving angiogenesis and bioenergetics in hyperglycemia.     

Perfusion cultures of human tumor cells: a scalable production platform for oncolytic adenoviral vectors

This study demonstrates the novel use of the HeLaS3 human tumor cell line for propagating ONYX-411, a recombinant oncolytic adenoviral vector. HeLaS3 cells enabled high levels of vector production without the risk of generating vector recombinants, which is possible with HEK293 cells. The development of a high-cell-density perfusion process using ATF technology yielded production levels as high as 6 x 10(11) vp/mL, which was approximately sevenfold greater than the titers achieved in fed-batch bioreactors. Several experiments were performed at the bench (15 L) and pilot (70 L) scales to demonstrate the robust and scalable nature of this industrially relevant technology.     

Adaptation of Escherichia coli O157:H7 to pH alters membrane lipid composition, verotoxin secretion, and resistance to simulated gastric fluid acid

The influence of adaptation to pH (from pH 5.0 to 9.0) on membrane lipid composition, verotoxin concentration, and resistance to acidic conditions in simulated gastric fluid (SGF) (pH 1.5, 37 degrees C) was determined for Escherichia coli O157:H7 (HEC, ATCC 43895), an rpoS-deficient mutant of ATCC 43895 (HEC-RM, FRIK 816-3), and nonpathogenic E. coli (NPEC, ATCC 25922). Regardless of the strain, D values (in SGF) of acid-adapted cells were higher than those of non-acid-adapted cells, with HEC adapted at pH 5.0 having the greatest D value, i.e., 25.6 min. Acid adaptation increased the amounts of palmitic acid (C16:0) and decreased cis-vaccenic acid (C18:1 omega 7c) in the membrane lipids of all strains. The ratio of cis-vaccenic acid to palmitic acid increased at acidic pH, causing a decrease in membrane fluidity. HEC adapted to pH 8.3 and HEC-RM adapted to pH 7.3 exhibited the greatest verotoxin concentrations (2,470 and 1,460 ng/ml, respectively) at approximately 10(8) CFU/ml. In addition, the ratio of extracellular to intracellular verotoxin concentration decreased at acidic pH, possibly due to the decrease of membrane fluidity. These results suggest that while the rpoS gene does not influence acid resistance in acid-adapted cells it does confer decreased membrane fluidity, which may increase acid resistance and decrease verotoxin secretion.     

Investigation of selective protein immobilization on charged protein array by wavelength interrogation-based SPR sensor

We have investigated the use of multilayer films of polyelectrolytes as selective surfaces to analyze protein interactions with a self-assembled SPR wavelength-shift sensor. Charged arrays were prepared by alternating adsorption of the charged polyelectrolytes, poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS). Multilayer formation was monitored with the SPR wavelength-shift sensor and a Spreeta SPR sensor. Protein immobilization on the charged surfaces, which was also analyzed by the SPR sensors, was dependent on the pI of the proteins. Tissue transglutaminase (tTGase) and beta-galactosidase (pIs, 5.1 and 5.3, respectively) were preferentially bound to the positively charged PDDA surface, whereas lysozyme (pI, 11.0) was selectively bound to the negatively charged PSS surface. Immobilization of tTGase on the PDDA surface was also dependent on the buffer pH. The interaction of tTGase with RhoA(V14), a constitutively active form of Rho, could be detected on the charged arrays with the wavelength-shift sensor. The arrays could be reutilized at least 5 times. Thus, it is likely that charged surfaces, assembled by the layer-by-layer method using polyelectrolytes, will prove useful for preparing selective protein arrays.     

Recombinant goldfish growth hormones (gfGH-I and -II) expressed in Escherichia coli have similar biological activities

Complementary DNA regions coding for two different mature goldfish growth hormones (gfGH-I and gfGH-II) with four and five cysteine residues were cloned into the bacterial expression vector, pRSETA. The recombinant gfGH-I (five cysteines) and -II (four cysteines) were produced in Escherichia coli as the fusion proteins carrying N-terminal 6XHis tag, which facilitates purification by using metal chelating affinity chromatography under denaturing condition with urea. The recombinant hormones were further refolded by gradually removing the urea. Native gfGH was also purified from goldfish pituitary glands and served as a positive control in the present study. The native and recombinant hormones were tested in goldfish hepatic radioreceptor assay and in vitro Spi 2.1 promoter activation assay. Our results showed that the two recombinant gfGHs are biologically active, and they have similar biological activities despite their having different cysteine contents.     

Selective release of calpain produced alphalI-spectrin (alpha-fodrin) breakdown products by acute neuronal cell death

Activation of calpain results in the breakdown of alpha II spectrin (alpha-fodrin), a neuronal cytoskeleton protein, which has previously been detected in various in vitro and in vivo neuronal injury models. In this study, a 150 kDa spectrin breakdown product (SBDP150) was found to be released into the cell-conditioned media from SH-SY5Y cells treated with the calcium channel opener maitotoxin (MTX). SBDP150 release can be readily quantified on immunoblot using an SBDP150-specific polyclonal antibody. Increase of SBDP150 also correlated with cell death in a time-dependent manner. MDL28170, a selective calpain inhibitor, was the only protease inhibitor tested that significantly reduced MTX-induced SBDP150 release. The cell-conditioned media of cerebellar granule neurons challenged with excitotoxins (NMDA and kainate) also exhibited a significant increase of SBDP150 that was attenuated by pretreatment with an NMDA receptor antagonist, R(-)-3-(2-carbopiperazine-4-yl)-propyl-1-phosphonic acid (CPP), and MDL28170. In addition, hypoxic/hypoglycemic challenge of cerebrocortical cultures also resulted in SBDP150 liberation into the media. These results support the theory that an antibody-based detection of SBDP150 in the cell-conditioned media can be utilized to quantify injury to neural cells. Furthermore, SBDP150 may potentially be used as a surrogate biomarker for acute neuronal injury in clinical settings.     

Unusual pattern of bacterial ice nucleation gene evolution

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of 14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina- bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16S data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.      

β-Decay and the origin of optical purity

Improving Keszthelyis simple model the evolutionary appearance of concentration difference of enanthiomeric compounds due to their differential decomposition by -rays is investigated taking into account the racemization as well. It is shown that if the difference in the cross sections is very small then the resulting concentration difference will never exceed the statistical fluctuations, while in the case of a sufficiently large difference in the cross sections the concentration difference can overgrow the statistical fluctuations in an evolutionary reasonable period of time. The relative difference of the concentrations, however, will be generally much smaller than that of the cross sections. Therefore some other, amplifying mechanism must be postulated in order to explain the optical purity of living beings.