Different protective roles in vitro of alpha- and beta-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals.

It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.     

The C termini of Arabidopsis cryptochromes mediate a constitutive light response

Cryptochrome blue light photoreceptors share sequence similarity to photolyases, flavoproteins that mediate light-dependent DNA repair. However, cryptochromes lack photolyase activity and are characterized by distinguishing C-terminal domains. Here we show that the signaling mechanism of Arabidopsis cryptochrome is mediated through the C terminus. On fusion with beta-glucuronidase (GUS), both the Arabidopsis CRY1 C-terminal domain (CCT1) and the CRY2 C-terminal domain (CCT2) mediate a constitutive light response. This constitutive photomorphogenic (COP) phenotype was not observed for mutants of cct1 corresponding to previously described cry1 alleles. We propose that the C-terminal domain of Arabidopsis cryptochrome is maintained in an inactive state in the dark. Irradiation with blue light relieves this repression, presumably through an intra- or intermolecular redox reaction mediated through the flavin bound to the N-terminal photolyase-like domain.     

Apolipoprotein A-II/A-I ratio is a key determinant in vivo of HDL concentration and formation of pre-beta HDL containing apolipoprotein A-II

Overexpression of human apolipoprotein A-II (apo A-II) in mice induced postprandial hypertriglyceridemia and marked reduction in plasma HDL concentration and particle size [Boisfer et al. (1999) J. Biol. Chem. 274, 11564-11572]. We presently compared lipoprotein metabolism in three transgenic lines displaying plasma concentrations of human apo A-II ranging from normal to 4 times higher, under ad libitum feeding and after an overnight fast. Fasting dramatically decreased VLDL and lowered circulating human apo A-II in transgenic mice; conversely, plasma HDL levels increased in all genotypes. The apo A-I content of HDL was inversely related to the expression of human apo A-II, probably reflecting displacement of apo A-I by an excess of apo A-II. Thus, the molar ratios of apo A-II/A-I in HDL were significantly higher in fed as compared with fasted animals of the same transgenic line, while endogenous LCAT activity concomitantly decreased. The number and size of HDL particles decreased in direct proportion to the level of human apo A-II expression. Apo A-II was abundantly present in all HDL particles, in contrast to apo A-I mainly present in large ones. Two novel findings were the presence of pre-beta migrating HDL transporting only human apo A-II in the higher-expressing mice and the increase of plasma HDL concentrations by fasting in control and transgenic mice. These findings highlight the reciprocal modifications of VLDL and HDL induced by the feeding-fasting transition and the key role of the molar ratio of apo A-II/A-I as a determinant of HDL particle metabolism and pre-beta HDL formation.     

Stress-induced RNASET2 overexpression mediates melanocyte apoptosis via the TRAF2 pathway in vitro

The recent genome-wide association study identified a link between vitiligo and genetic variants in the ribonuclease T2 (RNASET2) gene; however, the functional roles of RNASET2 in vitiligo pathogenesis or in melanocyte apoptosis have yet to be determined. The current study was designed to investigate the vitiligo-related expression pattern of RNASET2 and its molecular function involving apoptosis-related signaling proteins and pathways. The results showed overexpression of RNASET2 in epidermis specimens from 40 vitiligo patients compared with that from matched healthy controls. In addition, in vitro analyses indicated that overexpression of RNASET2 was inducible in cultured primary human melanocytes and keratinocytes by stress conditions, that is, exposure to UV irradiation, hydrogen peroxide, and inflammatory factors, respectively, and led to increased cell apoptosis via the tumor necrosis factor receptor-associated factor 2 (TRAF2)–caspases pathway through the physical interaction of RNASET2 with TRAF2. Thus, RNASET2 may contribute to vitiligo pathogenesis by inhibiting TRAF2 expression and, as such, RNASET2 may represent a potential therapeutic target of vitiligo.     

内皮素—1预处理大鼠心脏Gaq／11和Gia蛋白含量的变化

The present study was undertaken to explore the mechanism of G protein-mediated signal transduction pathway during endothelin-1 (ET-1) pre-treatment and ischemic preconditioning (IP). Rats were divided into four groups: ET-1, IP, ischaemia-reperfusion (IR) and control groups. ET-1 pre-treatment model was prepared by administrating 0.5 nmol/(L.kg) ET-1 into rat left ventricle, whereas IP model was prepared by ligating the left coronary artery for 5 min followed by 30 min reperfusion. All the animals were subjected to 60 min regional ischaemia and 30 min reperfusion alternately and then parameters of ventricular arrhythmia and expression of cardiac Galphaq/11 and Gialpha2 were measured. The results showed that the scores of ventricular arrhythmia decreased significantly in both ET-1 and IP treated groups as compared with IR group. In comparison with control group, Galphaq/11 increased by 77.8% (P<0.05) and 110.6% (P<0.01) in IP and ET-1 group respectively. Gialpha2 showed no significant difference in IP group, while it decreased by 31.0% (P<0.01) in ET-1 group. In conclusion, activation of G alphaq/11 may be related to the protecting mechanism of ET-1 pre-treatment and IP, whereas Gialpha2 may only play a role in ET-1 pre-treatment.     

Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1

Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo.     

Extracellular expression, purification, and characterization of a winter flounder antifreeze polypeptide from Escherichia coli

HPLC6 is the major component of liver-type antifreeze polypeptides (AFPs) from the winter flounder, Pleuronectes americanus. To facilitate mutagenesis studies of this protein, a gene encoding the 37-amino acid mature polypeptide was chemically synthesized and cloned into the Tac cassette immediately after the bacterial ompA leader sequence for direct excretion of the AFP into the culture medium. Escherichia coli transformant with the construct placIQpar8AF was cultured in M9 medium. The recombinant AFP (rAFP) was detected by a competitive enzyme-linked immunosorbent assay (ELISA). After IPTG induction, a biologically active rAFP was expressed. The majority of the rAFP was excreted into the culture medium with only trace amounts trapped in the periplasmic space and cytoplasm. After 18 h of induction, the accumulated rAFP in the culture medium amounted to about 16 mg/L. The excreted AFP was purified from the culture medium by a single-step reverse-phase HPLC. Mass spectrometric and amino acid composition analyses confirmed the identity of the purified product. The rAFP, which lacked amidation at the C-terminal, was about 70% active when compared to the amidated wild-type protein, thus confirming the importance of C-terminal cap structure in protein stability and function.